Vol. 5, 637-646, June 1994 Cell Growth & Differentiation 637

Dysfunction of the Myc-induced Apoptosis MechanismAccompanies c-myc Activation in the

. . . 1Tumorigenic 1929 Cell Line

Linda M. Facchini, Shaojun Chen, and Linda J. Z. Penn2

Departments of Microbiology, Immunology and Cancer,Hospital for Sick Children and Research Institute EL. M. F., S. C.,L. i. z. p.], and University of Toronto EL. M. F., L. J. z. P.1,Toronto, Ontario M5G 1 X8, Canada

Abstrad

Adivation of the c-myc protooncogene, resulting inderegulated, over-expression of the c-Myc protein, caninduce both cell proliferation and programmed celldeath (apoptosis) in nontransformed cells. Yet, c-mycadivation is commonly tolerated in many tumors. Thisapparent paradox can be resolved if adivation of c-mycin transformed cells is associated with loss of Myc-induced apoptosis. To examine this hypothesis, wecharaderized both the mechanisms of c-myc adivationand programmed cell death in the tumorigenic L929 cellline. We showed that adivation of c-myc in the 1929cell line involves several distind mechanisms, includingdysfundion of the Myc autosuppression pathway andalteration of c-Myc protein expression. In addition, wedemonstrated that 1929 cells do not undergo Myc-induced apoptosis. Analysis of somatic cell hybridsrevealed that this abrogation of programmed cell deathcan be partially restored and is likely due to one ormore genetic lesions. Our results support the hypothesisthat the dysfundion of the Myc-induced apoptosismechanism can accompany c-myc adivation andprovide an in vivo example illustrating two cooperativeevents which can contribute to tumor progression.

Introdudion

The c-myc protooncogene encodes a nuclear phosphopro-tein which is ubiquitously expressed in all cell types. A largebody ofexperimental evidence collectively shows that mul-tiple activities are attributable to the product of the c-mycgene (for reviews see Refs. 1-3). It appears that the c-Mycprotein plays a central role in the processes of cell prolif-eration, differentiation, immortalization, and programmedcell death. While the molecular mechanisms of these ac-tivities remain unclear, recent evidence strongly suggeststhat the c-Myc protein may elicit some or all ofthese eventsby functioning as a regulator of gene transcription (4-9).However, direct cellular gene targets for c-Myc have yet tobe identified.

Received 1/4/94; revised 3/24/94; accepted 3/31/94.

1 This work was supported by operating grants to L. J. z. P. from the National

Cancer Institute and the Medical Research Council of Canada. L. M. F. wassupported by studentships initially from the Hospital for Sick Children Re-search Institute and presently from the Medical Research Council of Canada.2 To whom requests for reprints should be addressed, at Departments ofMicrobiology, Immunology and Cancer, Hospital for Sick Children, 555University Avenue, Toronto, Ontario M5G 1 X8, Canada.

Regulated c-myc gene expression is critical for controlledcell proliferation, whereas deregulated, constitutive over-expression of c-myc is a frequent hallmark of tumor-derivedcells. Indeed, activation of the c-myc protooncogene cancontribute to the stepwise progression of tumorigenesis.Introduction of activated c-myc alone into primary cellsleads to immortalization, while introduction of c-myc and acooperating oncogene such as ras results in malignanttransformation (Refs. 2 and 1 0 and references therein). Sim-ilar effects are seen in animal models (1 i , 1 2). For example,in transgenic mice, expression of a c-myc transgene underthe control of a tissue-specific promoter leads to the devel-opment of tumors in the target tissue (1 3, 1 4). Thus, c-Mycis a strong potentiator of cellular proliferation.

Genetic alterations disrupting a normal regulatory mech-anism controlling c-myc expression may activate the c-mycprotooncogene to its oncogenic form. In a subset of tumors,c-myc activation can be attributed to gross structural alter-ations of the c-myc locus. These include retroviral trans-duction, promoter and enhancer element insertion, geneamplification, and chromosomal translocation (reviewed inRefs. 1 0 and i 5). However, the remaining tumors do notdemonstrate such structural alterations, indicating the exis-tence of alternative mechanisms of c-myc activation. Weand others have shown that primary cells as well as non-transformed, established cell lines possess a Myc negativefeedback mechanism, whereby Myc protein suppressestranscription initiation from the c-myc promoter (1 6-19).Furthermore, loss of this mechanism may deregulate c-myc

expression and contribute to the process of cellular trans-formation, as many human tumorigenic cell lines no longerdemonstrate Myc negative autoregulation (1 7). Indeed, wehave demonstrated, through somatic cell hybridizations be-tween autosuppression-competent and dysfunctional fibro-blast cell lines, that c-myc activation can result from thedysfunction of one or more trans-acting factors required forMyc autosuppression (18).

As mentioned above, the c-Myc protein likely performsseveral cellular functions. It was recently demonstrated thatc-Myc expression, under growth-restricting conditions, canoperate in a dose-dependent manner to induce apoptosis(20-22). This programmed cell death is a normal and anessential biological feature in the development and main-tenance of cells in multicellular organisms and differsfrom trauma-induced necrosis. While a variety of stimulican trigger apoptosis in various cell types, all apoptoticcells exhibit similar morphological changes including thedevelopment of pyknotic nuclei, membrane blebbing,extensive chromatin condensation, and DNA fragmenta-tion (for reviews see Refs. 23-25). Although it is likelythat programmed cell death is a genetically based proc-ess (26-29), the molecular components of apoptosis,including the mechanism of Myc-induced apoptosis, re-main largely unknown.

Ifderegulated c-Myc protein expression induces both cellproliferation and cell death, then how is it that tumor cells

a b

II II

Rat c-myc -*

Mousec-myc �

Li��f..

� .�

(;APDH -.�-

++

� v-gag-m3

�.

Fig. 1. Myc negative autoregulation is dysfunctional in the L929 cell line.

RNase protection analysis of 10 pg RNA from subconfluent Rat-i and L929fibroblasts infected with a control retrovirus )-, DORneor and pBabehygror,

respectively) or a retrovirus carrying the viral gag-myc gene (+, pDoKv-myd

nec! and pBabev-myc/hygrcf, respectively). a, expression of endogenous ratand mouse c-myc was detected using a probe to rat c-myc exon I gene

sequences. Both rat and murine GAPDH expression were detected with aprobe to the mouse GAPDH gene. The unlabeled band in the L929 (-) lanecorresponds to excess, undigested GAPDH probe. b, ectopic expression of

v-gag-myc was detected with a probe to the viral gag sequences of the avianmyelocytomatosis virus MC29. The rat c-myc exon I and v-gag-myc probes

do not hybridize with v-myc and c-myc, respectively.

638 c-niyc Activation, Autoregulation, and Apoptosis

tolerate an activated c-myc gene? One plausible explana-tion suggests that during the process of transformation, ac-tivation of c-myc must be accompanied by inhibition of theMyc-induced apoptotic pathway (21, 30, 31). To addressthis possibility, we explored the transformation mechanismof the tumonigenic L929 cell line. We show in this paperthat both c-myc gene and c-Myc protein expression arederegulated in L929 cells. Moreover, as proposed, Myc-induced apoptosis is not demonstrable in Myc-activated

L929 cells. In addition, somatic cell hybridization expeni-ments show that this resistance to Myc-induced apoptosiscan be overcome. These results suggest that the transfor-mation of the tumonigenic L929 cell line involved both theactivation of the c-myc oncogene and inhibition of theMyc-induced apoptosis pathway.

Results

Dysfunction of the Myc Negative Autoregulation Mecha-nism Contributes to Deregulated c-myc Expression in 1929Cells. To examine the integrity of the Myc negative feed-back mechanism, L929 cells were infected with either areplication-deficient control retnovi nus carrying the neomy-cm drug resistance (ned) gene or a netnovinus carrying viralgag-myc (v-myc) as well as the neor genes. Retroviral in-fection results in the stable integration of a single copy ofthe retnovinal genome into each target cell. While enforcedexpression of either v-Myc on c-Myc protein can reduceendogenous c-myc gene expression (i 8), in these studiesv-Myc was used as the effector Myc molecule to readilydistinguish ectopic v-myc from endogenous c-myc expres-sion within the cell. Cells were selected in medium con-taming G418 sulfate, and drug-resistant colonies werepooled. Total cellular RNA was harvested from subconflu-ent, proliferating cells and assayed by RNase protection.Endogenous c-myc RNA levels were detected by an anti-sense RNA probe derived from the 5’ end ofexon I ofthe ratc-myc gene. This probe protects 500- and 350-base, P1-

and P2-initiated homologous nat c-myc RNA fragments andi 20- and 90-base heterologous munine c-myc RNA frag-ments. The probe does not hybridize to the v-myc RNAbecause v-myc lacks exon I sequences. In all RNase pro-

tections described, the expression patterns of P1 - and P2-initiated rat c-myc fragments are similar; however, only themore abundant P2 fragment is shown. Levels of ectopicexpression of v-myc were determined using an antisenseRNA probe to the viral gag sequences of the v-myc gene,which generates a i 50-base protected fragment.

The basal level of endogenous munine c-myc RNA wasreadily detected in L929 cells infected with control retro-virus and remained virtually unchanged in L929 cells in-fected with netnovirus expressing v-myc (Fig. 1 a). The levelof ectopic v-myc RNA expression in the L929 cells was notlimiting and was greaten than the level of ectopic v-mycRNA expression required to elicit the Myc autosuppressionmechanism in feedback-competent Rat-i cells (Fig. 1, aand b). Thus, L929 cells possess a dysfunction in the Mycnegative autoregulatory pathway, resulting in deregulatedc-myc RNA expression.

The Myc Negative Feedback Mechanism Is Dysfunc-tional in 1929 Cells Due to Multiple Mutations. Weshowed previously that somatic cell hybridization to feed-back-competent Rat-i cells restores Myc negative autoneg-ulatony activity in an autosuppression-dysfunctional NIH3T3 cell line (18). This complementation cleanly demon-

strated that the dysfunction in the NIH 3T3 cells was me-diated by trans-acting factors. To determine the nature ofthe Myc negative autoregulation dysfunction in L929 cells,a similar approach was used to generate somatic cell hy-bnids between the L929 and Rat-i cell lines. The two pa-rental cell lines, each expressing a different drug resistancegene, were densely coseeded and fused by polyethyleneglycol treatment. Somatic cell hybrids were selected inculture medium containing both drugs, and individualdrug-resistant colonies were ring cloned and expanded.RNA was harvested from each clone and was subsequentlyassayed by RNase protection to determine the levels ofendogenous rat and munine c-myc as well as ectopic v-mycexpression.

a Rat-i L929x xRat-i L929 Rat-i x L929

I ii �I I

- -++- -++ --..++++

Rat c-myc .-i.i..� ‘ -- �

r_#{248}�#{149}�_m �� .1

Mousec-myc

� �-

GAPDH � #{149}�.4#{248}�#{149}4��4i4#{216}�

I 2 3 4 5 6 7 8 910111213141516

b Rat-i L929 Rat-ix x x

Rat-i L929 L929II II I �i

+ ++ + + +++

v-gag-myc -�- .rn#{248}’��’�

Cell Growth & Differentiation 639

Fig. 2. The autosuppression dysfunction in 1929 cells is due to multiplemutations. RNA from control somatic cell hybrid clones (-) and from somatic

To control for the drug selection conditions, each paren-tal cell line was hybridized to itself so that the hybrid cellsexpressed one ofthe hygnomycin resistance (hygror) or neorgenes. As expected, none of the parental hybrids survivedfollowing selection in both hygromycin B and G4i 8 sulfate(data not shown). Additional control hybridizations wereperformed to determine whether: (a) the hybridization pro-cedure itself may affect the mechanism of Myc negativeautoregulation; and (b) an increased level of ectopic v-mycexpression may affect the capacity of the parental cell lineto demonstrate Myc autosuppression. These control hybrid-izations included Rat-i neor x Rat-i hygror, Rat-i v-myc/neor x Rat-i v�myc/hygror, L929 neor x L929 hygror,and L929 v�myc/neor x L929 v�myc/hygror. Rat c-myc

RNA was readily detectable in the control Rat-i X Rat-ihybrids and was suppressed in the Rat-i x Rat-i hybridsexpressing v-myc (Fig. 2a, Lanes 1 to 4). In contrast, basallevels of munine c-myc RNA in the L929 X L929 hybridsshowed no appreciable change between the control cells

and cells expressing v-myc (Fig. 2a, Lanes 5 to 8). Theresults of these hybridization controls demonstrated that

both the hybridization procedure and additional ectopicv-myc expression did not alter either the ability of Rat-i

on the inability of L929 cells to demonstrate a Myc neg-

ative feedback response.The experimental hybridizations involved the following

two cell fusions: (a) to establish basal levels of both rat andmunine c-myc RNA in the hybrids, a Rat-i neor x L929hygror fusion was conducted; and (b), to determine whether

the Myc autosuppression dysfunction in L929 cells could becomplemented by trans-acting rat cell factors or remainedrefractory to complementation, suggesting the involvementof cis-acting mutations, a Rat-i v�myc/neor x L929 v-myc/hygror fusion was performed.

In the aforementioned somatic cell hybridizations be-

tween autosuppression-competent Rat-i and dysfunctionalNIH 3T3 cell lines, both rat and munine c-myc RNA levelswere fully suppressed in hybrids expressing v-myc relativeto basal levels of c-myc expression in control hybrids (18).Complete restoration of Myc negative autoregulatory activ-ity at the munine locus unequivocally established that trans-acting factors were responsible for dysfunction of Myc au-tosuppression in the NIH 3T3 cell line. The results of theRat-i X L929 hybridization revealed a complex and distinctpattern of c-myc expression. Specifically, as in the previousRat-i X NIH 3T3 hybridizations, rat c-myc RNA levels were

suppressed in the Rat-i x L929 hybrids expressing v-mycrelative to basal rat c-myc RNA levels in the control Rat-i XL929 hybrids (Fig. 2a; compare rat c-myc bands in Lanes 13to 16 to Lanes 9 to 12). However, the steady-state level ofmunine c-myc RNA was not significantly non consistently

suppressed in the Rat-i x L929 hybrids expressing v-myc

cell hybrid clones expressing v-gag-myc (+) was analyzed by RNase pro-tection to detect expression levels of (a) endogenous rat and mouse c-myc

genes and (b) the v-gag-myc gene. For rat c-myc, only P2-initiated protectedRNA fragments are shown. Unlabeled bands correspond to excess, undi-gested GAPDH probe. The following somatic cell hybridizations were per-

... . .- . � � . formed: a, Rat-i ned X Rat-i hygro’ (Lanes 1 and 2); Rat-i v-myc/neo’ xGAPDH -� 4�P#{149}�*II�. Rat-i v-myc/hygrd (Lanes 3 and 4); L929 ned x L929 hygro’ (Lanes 5 and

1 2 3 4 5 6 7 8 6); L929 v-myc/neo’ x 1929 v-mydhygro’ (Lanes 7 and 8); Rat-i ned X

L929 hygro’ (Lanes 9 to 12); Rat-i v-myc/neo’ x L929 v-myc/hygro’ (Lanes

13 to 16); b, Rat-i v-myc/neo’ X Rat-i v-myc/hygro’ (Lanes 1 and 2); L929

v�myc/neor x L929 v-myc/hygro’ (Lanes 3 and 4); Rat-i v-myc/neo’ X L929v-myc/hygro’ (Lanes 5 to 8).

205 -

116 -

80-7

49 -

1 2 3 4

Fig. 3. Elevation of Myc protein concentration in L929 cells. Immunoblot

analysis of Myc protein levels in whole cell extracts from iO� (Lane 1) andi o� (Lane 2) HL-60, 5 X 1 o� Rat-i (Lane 3), and 5 X i O� L929 (Lane 4) cells.

Total cellular protein was resolved on a iO% sodium dodecyl sulfate-

polyacrylamide gel and transferred to nitrocellulose membrane. Myc protein,indicated by arrows to the p64 and p67 species, was detected using poly-

clonal pan-Myc rabbit antibody and visualized by enhanced chemilumines-

cence. Size markers correspond to prestained high range standards.

640 c-rnyc Activation, Autoregulation, and Apoptosis

3 The abbreviation used is: FBS, fetal bovine serum.

compared with basal munine c-myc RNA levels in controlRat-i X L929 hybrids (Fig. 2a; compare mouse c-myc bandsin Lanes 13 to 16 to Lanes 9 to 12). Thus, while the Mycautosuppression mechanism was functional in the Rat-i XL929 hybrids, as shown by suppression of endogenous ratc-myc RNA, trans-acting nat cell components were unableto complement the L929 autosuppression dysfunction andfully down-regulate endogenous munine c-myc expression.Interestingly, there was a substantial and unexpected de-crease in basal rat c-myc RNA levels in the control Rat-i XL929 hybrids compared to basal rat c-myc RNA levelsdetected in Rat-i x Rat-i control hybrids or the Rat-iparental cell line [compare rat c-myc bands in Fig. 2a,Lanes 9 to 12 to Fig. 2a, Lanes 1 and 2 and Fig. iaRat-i (-)]. These results suggested that components of theL929 cells exert a trans-dominant effect on basal rat c-myc

expression.In the hybridizations described above, ectopic expres-

sion of the v-myc gene was analyzed by the RNaseprotection assay. The levels of v-myc RNA in the Rat-iv-myc X L929 v-myc hybrids were consistently lowerthan levels detected in the Rat-i v-myc x Rat-i v-mycand L929 v-myc x L929 v-myc counterparts (Fig. 2b;

compare Lanes 5 to 8 to Lanes 1 to 4). This pattern ofexpression suggested that higher levels of v-myc expres-sion are not tolerated in the Rat-i x L929 hybrids.

c-Myc Protein Expression Is Altered in 1929 Cells Corn-pared to Rat-i Cells. Another intriguing observation fromthe above experiments is the suppression of basal level ratc-myc RNA in control Rat-i X L929 somatic cell hybrids inthe absence of ectopic v-myc expression. Conceivably, anelevated level of endogenous munine c-Myc protein, con-tributed by the L929 parental cells, could be invoking theMyc autosuppression mechanism in the hybrids. To test thishypothesis, the relative abundance of Myc protein in Rat-iand L929 parental cell lines was determined by immuno-blot analysis. Whole cell extracts from subconfluent, pro-liferating cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotnansferredto nitrocellulose membrane; total c-Myc protein was de-tected using polyclonal pan-Myc antiserum (a kind gift of G.Evan, Imperial Cancer Research Fund, London, U.K.). A lowbasal level of c-Myc protein was detected in Rat-i cells (Fig.3, Lane 3), consistent with previous reports (i8, i9). Incontrast, L929 cells demonstrated a high basal level ofc-Myc protein (Fig. 3, Lane 4). Interestingly, the ratio ofp64/p67 c-Myc protein species was approximately equal inthe L929 cells, while in Rat-i fibroblasts, the p64 proteinwas the major detectable species of c-Myc. To quantitatethese Myc protein levels, the immunoblot autoradiognamswere scanned by densitometny because the Myc enzyme-linked immunosorbence assay can not detect c-Myc proteinin Rat-i cells (i 8). The number of c-Myc molecules/cell wasestimated by comparing the Rat-i and L929 c-Myc proteinlevels to c-Myc levels found in HL-60 cells (Fig. 3) in whichthe concentration of c-Myc protein has been establishedpreviously by a Myc enzyme-linked immunosorbence assay(32). By this approach, we estimated that sub-confluent,proliferating Rat-i cells express approximately 800 c-Mycmolecules/cell, while proliferating L929 cells express ap-proximately 6500 c-Myc molecules/cell, representing an8-fold increase in c-Myc protein levels (data not shown). Asthis 8-fold induction in Myc protein expression is not re-flected in the c-myc RNA levels of L929 cells (Fig. 2a), it isintriguing to speculate that the abundance of c-Myc protein

Cl4�, 4,)

� �

in these cells is the result of deregulation at the translationallevel.

Myc-induced Apoptosis Is Not Observed in 1929 Cells.Myc protein in combination with a block to proliferationcan induce the apoptotic pathway in primary and immon-talized rat fibroblasts (21, 33), T-cell hybnidomas (22),and IL-3-dependent myeloid cells (20). To examine theMyc-induced apoptotic response of L929 v-myc cellsunder conditions of serum deprivation, duplicate culturesof control Rat-i and Rat-i v-myc cells and control L929and L929 v-myc cells were grown to confluence inmedium containing i0% FBS.3 After 2 days, one set ofcultures was transferred to medium containing 0.1 % FBS,and cell deaths were monitored by microscopy. To dis-tinguish between apoptosis and trauma-induced necrosisin those cell lines which exhibited death events, DNAwas extracted from nonadherent cells and analyzed forchromatin fragmentation.

As reported previously (21), Rat-i v-myc cells demon-strated extensive cell death within 24 h following serumwithdrawal (Fig. 48), and the degree ofcell death continuedto escalate after 4 days of serum deprivation (Fig. 4D).Interestingly,the control Rat-i cells maintained in low se-rum medium also experienced a small but detectable num-ben of cell deaths after 24 h (Fig. 4A); however, evidence ofdeath events decreased thereafter and completely disap-peared after 4 days (Fig. 4C). Qualitative analysis of DNAharvested from total nonadherent cells indicated a chroma-tin fragmentation pattern characteristic of apoptotic celldeath (Fig. 5a, Lanes 1 and 2, and data not shown). Thus,serum-deprived Rat-i fibroblasts appear to undergo an mi-tial wave of apoptosis, which subsides in cells expressing anormal c-myc gene and intensifies in those cells expressingan activated myc gene.

Rat-I L929 Rat-i x L929

Fig. 4. Myc-induced apoptosis is

dysfunctional in L929 cells and re-suIts from multiple genetic lesions.Polaroid photomicrographs of con-

fluent cell monolayers maintainedin culture medium supplemented

with 0.1 % FBS for 1 day (A, B, E, F,

I,and I) or 4 days (C, 0, C, H, K,

and L). The extent of cell death inRat-i and L929 fibroblasts infected

with a control retrovirus (A, C, E,

and C) was compared to Rat-i andL929 fibroblasts infected with ret-rovirus expressing v-myc (B, D, F,

and H). The control somatic cell

hybrid clone (I and K) was com-pared to the hybrid clone express-ing v-myc (I and L) and to the ro-dent fibroblasts. One of four

randomly chosen clones is shown

for both the control hybrids andhybrids expressing v-myc. Rat-icontrol, day 1 (A); Rat-i v-myc,

day i (B); Rat-i control, day 4 (C);Rat-i v-myc, day 4 (0); L929 con-

trol, day i (E); L929 v-myc, day i(F); L929 control, day 4 (C); L929v-myc, day 4 (H); Rat-i X L929

control, day i (I); Rat-i v-myc XL929 v-myc, day 1 (1); Rat-i x

L929 control, day 4 (K); Rat-iv-myc x L929 v-myc, day 4 (L).x 200.

F� ‘� � � � :� . , ,� . , � � �, , :� � � �‘

k� � ‘ “ #{149}� #{149}..�� �.:: :

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: � � � � � � � � � � �, .. �..

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Cell Growth & Differentiation 641

j�*�

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In contrast, evidence of cell death was not seen in eithergrowth-arrested control L929 or L929 v-myc cell culturesafter 1 , 4, or 7 days of serum deprivation (Fig. 4, E, F, C, andH; and data not shown). While control cultures propagatedin medium containing iO% FBS exhibited extensive pilingof cells, indicating a loss of contact inhibition (data notshown), all cultures maintained in low serum medium didnot exhibit cell piling but instead formed dense monolayers.The density of the monolayer reached a maximum levelafter 2 to 3 days in medium containing 0.i% FBS andremained constant thereafter throughout the 7-day period(data not shown), indicating that the serum-deprived L929cells were growth arrested. Therefore, L929 cells do notdemonstrate Myc-induced apoptotic activity when a prolif-eration block is imposed.

Lack of Myc-induced Apoptosis in [929 Cells Can BeComplemented by Somatic Cell Hybridization. To gaininsight into the nature of the lesions in the Myc-inducedprogrammed cell death pathway in L929 cells, we exam-ned Rat-i neor x L929 hygror and Rat-i v�myc/neor x

L929 v�myc/hygror somatic cell hybrid clones for the in-duction of apoptosis upon growth arrest by serum depniva-tion. Morphologically, the hybrid clones exhibited a phe-

notype intermediate to Rat-i and L929 cells (Fig. 4:compare /to A and F; Kto Cand G). Specifically, the cellswere akin to Rat-i fibroblasts in general size and shape butwere more refractile, like L929 cells. In addition, whenpropagated in medium containing i 0% FBS, the hybridsdisplayed the contact inhibition seen in the rat fibroblasts(data not shown).

Upon transfer to medium containing 0.i % FBS, three offour control Rat-i x L929 hybrid clones demonstrated amodest amount of cell death after 24 h (Fig. 4!). This celldeath was due to apoptosis, as confirmed by DNA fragmen-tation (Fig. 5b, Lanes 1 to 4) and subsided completely after4 days of serum deprivation (Fig. 4K), reminiscent of thewave of apoptosis seen in the control Rat-i parental cells(Fig. 4, A and C).

All four Rat-i v-myc X L929 v-myc hybrid clones dis-played apoptosis after 24 h of serum deprivation (Fig. 4);Fig. Sb, Lanes 5-8). The extent of cell death was greaterthan that observed in the control Rat-i x L929 hybridclones (Fig. 4!) but less than that seen in the Rat-i v-mycparental cell line (Fig. 48). In addition, the degree ofapoptosis did not escalate with prolonged exposure tolow serum medium, as was the case in the Rat-i v-myc

aM i 2 3 4

b

I” 1234 567 8

Fig. 5. Cell death in Rat-i fibroblasts and Rat-i x L929 somatic cell hybridsis due to apoptosis. Nucleosomal fragmentation analysis of DNA extractedfrom detached cells after culture of confluent monolayer for i day in medium

supplemented with 0. 1 ‘Y�, FBS. a, 1.5/, agarose gel with 1 00-base pair ladderDNA marker )M); Rat-i control (Lane 1); Rat-i v-myc)Lane2); L929 control(Lane 3); L929 v-rnyc (Lane 4(. h, 1 .0/, agarose-2.0”/ NuSieve gel with100-base pair ladder DNA marker (M); Rat-i X L929 control (Lanes 1 to 4);Rat-i v-rnyc X L929 v-myc (Lanes 5 to 8).

642 c-myc Activation, Autoregulation, and Apoptosis

Recent work with c-myc and bc/-2 genes has suggested anovel mechanism of oncogene collaboration in which on-

Cells (Fig. 4D), but slowed substantially after 4 days (Fig.4L) and subsided completely after 7 days of serum de-privation (data not shown). The surviving cells were stillsensitive to MyC-induced apoptosis because a similartransient death phenotype was observed when two ofthese Clones were tested in a second, consecutive apop-tosis assay (data not shown). These results indicate thatdysfunction of the Myc-induced apoptosis mechanismcan be complemented through somatic cell hybridizationto an apoptosis-sensitive cell line.

Discussion

cogenic activation may synergize with a block in the pro-grammed cell death pathway to elicit cellular transforma-tion (30, 31). To explore this hypothesis, we havecharacterized the integrity of regulated c-myc expressionand the Myc-induced apoptosis mechanism in tumonigenicL929 cells. We have demonstrated that steady-state c-Mycprotein levels are elevated and that this deregulation ofc-Myc expression may involve abrogation of the Myc neg-ative autoregulation pathway as well as translational con-trol mechanisms. Moreover, we found that the Myc-in-duced programmed cell death pathway is not functional inthe L929 cell line. Interestingly, this lack of apoptosis can becomplemented by somatic cell hybridization to nontumoni-genic fibroblasts, suggesting that genetic lesions likely con-tribute to the apoptosis-resistant phenotype. The associationof c-myc activation with dysfunction ofthe apoptosis mech-anism in tumonigenic L929 cells represents an in vivo ex-ample of a novel form of oncogenic cooperation.

Mechanisms of c-myc Activation. Our previous somaticcell hybridization experiments showed that the lack of Mycautosuppnession in NIH 3T3 cells is mediated by a dysfunc-tion in one on more cellular trans-acting factors (1 8). Similarsomatic cell hybridizations between Myc autosuppression-competent Rat-i and dysfunctional L929 fibroblasts re-vealed a more complex pattern of c-myc deregulation. Sim-lan to Rat-i x NIH 3T3 hybrids, the Myc autosuppression

mechanism in the Rat-i X L929 hybrids was operative anddominant because endogenous rat c-myc RNA levels weresuppressed in response to both endogenous munine c-Mycprotein and ectopic v-myc gene expression. By contrast,levels of endogenous munine c-myc RNA in the Rat-i XL929 hybrid clones were not fully down-regulated in ne-sponse to v-myc expression. This result suggests that mul-tiple mutations in both ds and trans may be responsible forthe lack of Myc negative autoregulation in the L929 cells.One interpretation is that complementation of trans muta-tions by Rat-i cell factors resulted in full autosuppression ofone munine c-myc allele, whereas the second allele re-mained refractory to Myc negative autoregulation due to

cis-acting mutations. Southern blot analysis of DNA han-vested from L929 cells did not reveal any gross structuralalteration of the c-myc locus (data not shown). Therefore,these alterations in cis may include small deletions or pointmutations within the c-myc promoter on the transcriptionregulatory region required for autosuppression. Verificationand determination of the exact nature of these putativemutations will require direct sequence analysis and mayyield important clues to the mechanism of c-myc transcnip-tional autonegulation.

In general, c-Myc protein levels correlate with c-myc

RNA levels (reviewed in Ref. 1 5). For instance, both c-mycRNA and c-Myc protein levels demonstrate similar transientinduction patterns upon mitogen stimulation of quiescentcells (34, 35) and correlate during differentiation of mouseembryonal carcinoma cells (36). Interestingly, we havefound that steady-state C-myc RNA levels are comparablebetween proliferating Rat-i and L929 fibroblasts (Fig. 2;compare Lanes 1 and 2 to Lanes 5 and 6), and yet, L929cells show a significant 8-fold elevation in total c-Mycprotein compared to Rat-i cells (Fig. 3; compare Lanes 3and 4). The deregulated c-Myc protein in L929 cells isbiologically active because it is capable of participating inthe autosuppression of endogenous nat c-myc expression inthe control Rat-i x L929 hybrid cells (Fig. 2, Lanes 9-12).This additional mechanism of c-Myc activation in L929

Cell Growth & Differentiation 643

cells remains to be characterized but may be occurring atthe level of translational initiation, reflecting changes inprotein stability, or be due to other posttranslational mod-ifications. Therefore, c-Myc expression is deregulated in theL929 cell line by a potential translational activation mech-anism as well as disruption of the Myc autosuppressionmechanism.

Mechanisms of Myc-induced Programmed Cell Death.Under permissive growth conditions, activated c-myc willpotentiate cellular proliferation, yet the combination of anactivated c-myc gene with a block to cell cycle progressionproves to be deadly (21 , 30, 31 , 37). Therefore, undernonpermissive, physiological conditions in which growthfactor signaling is restricted, inhibition of the Myc-inducedapoptotic pathway is essential for successful completion ofc-myc-mediated cell proliferation (21 , 30, 31). However,the molecular mechanisms governing the induction, exe-cution, and inhibition of programmed cell death, includingMyc-i nduced apoptosis, are largely unknown.

Our L929 cells, which possess an activated c-myc genebut do not demonstrate Myc-induced apoptosis undergrowth-restricting conditions, support the above model.Conceivably, the abrogation of apoptosis in L929 cellscould result from loss-of-function mutations in the Myc-induced apoptotic pathway, other convergent apoptoticpathways, or from gain-of-function mutations invokingdominant inhibition of programmed cell death. To deter-mine which mechanism the L929 cell line has adopted totolerate the presence of deregulated c-Myc protein, weanalyzed Rat-i X L929 somatic cell hybrid clones for ap-optotic function. In this system, restoration of Myc-inducedapoptosis in the hybrids would suggest complementation ofa loss-of-function mutation by Rat-i cellular factors,whereas failure to do so would implicate activation ofgain-of-function lesions in a cellular survival or prolifera-tion pathway.

We found a transient vulnerability to Myc-induced ap-

optotic cell death in the somatic cell hybrids. The observa-tion that ectopic v-myc expression in the Rat-i v-myc XL929 v-myc hybrids is capable of inducing apoptosis (Fig.4]), supports the notion that L929 cells have acquired mu-tations which inactivate one or more essential genes incellular suicide pathways. Indeed, an excellent example ofsuch loss-of-function mutations occurs in vivo during on-togeny of Caenorhabditis e/egans (26). In the developingnematode, recessive mutations in the ced-3 or ced-4 genesprevent the initiation of the programmed cell death path-way in cells normally destined to die (Ref. 29 and referencestherein). In L929 cells, the inactivated genes may lie down-stream of c-Myc and function exclusively in the Myc-in-duced apoptosis mechanism, may overlap with other spe-cific apoptotic pathways, or may encode fundamentalcomponents required for the completion of apoptosis.

While restoration of Myc-induced apoptosis is evident inall four of the Rat-i v-myc x L929 v-myc hybrid clonesanalyzed, only a subpopulation of cells within each cloneis susceptible to or committed to programmed cell death.Unlike the Rat-i v-myc parental cell cultures, which dem-onstrate escalating numbers of death events upon pro-longed serum deprivation (Fig. 4D), the Rat-i v-myc X

L929 v-myc hybrid clones ultimately appear to successfullygrowth arrest (Fig. 4L). This phenomenon suggests the L929cells harbor mutations which actively inhibit programmedcell death and that this dominant inhibition occurs in adose-dependent manner to determine both the individual

hybrid cell fate and the overall phenotype of the culture.

Conceivably, this survival function could act indirectly tobypass the imposed proliferation block, independently ofc-Myc protein, to inhibit apoptosis induction or down-stream of c-Myc to rescue a cell not yet committed to death.One obvious candidate gene for this survival activity isbc/-2, which can inhibit apoptosis in a variety of cell types(38) including T-cells (39), Chinese hamster ovary cells (30),rat sympathetic neurons (40), and rat fibroblasts (3i). We

are presently exploring whether L929 cells express an ac-tivated bc/-2 oncogene. Thus, like Myc-induced prolifera-tion, Myc-induced apoptosis is a genetic mechanism in-volving genes which positively and negatively regulate theMyc-induced apoptotic pathway.

Like many proteins involved in growth control and de-velopmental regulation, c-Myc can function in a dose-de-pendent manner (2, i 8). Indeed, both the rate and degree ofinduction of apoptosis in rat fibroblasts depend upon thecellular concentrations of c-Myc protein (2i). The transientand limited nature of the apoptosis seen in both controlRat-i X L929 and Rat-i v-myc X L929 v-myc somatic cellhybrids could reflect total Myc protein concentration withinindividual cells. We suggest that within these populations,a small number of cells are sensitive to apoptosis as theresult of elevated Myc protein levels, while the majority ofcells escape commitment to apoptosis due to a balance ofMyc protein and survival factor activities.

Myc “Toxicity” in Nontransformed Cells. Immortalized,nontransformed cell lines, such as Rat-i fibroblasts, appearto be restricted in the maximum amount of Myc which canbe expressed (i 8), yet fully transformed, tumor-derived celllines such as HL-60 and HeLa exhibit significantly elevatedconcentrations of Myc protein (32). The results of our stud-ies are consistent with the notion that high Myc levels canbe fatal to nontransformed cells. Unlike Rat-i cells, L929cells can tolerate elevated Myc levels, possibly due to in-hibition of the Myc-induced apoptosis pathway. In Rat-iv-myc X L929 v-myc somatic cell hybrids, however, theprogrammed cell death pathway is functionally restored.Interestingly, the level of v-myc RNA in these hybrid clonesis consistently lower than that found in the control L929v-myc X L929 v-myc hybrids (Fig. 2b; compare Lanes 5 to8 to Lanes 3 and 4, respectively), suggesting that a selectionfor clones displaying a lower level of Myc expression hasoccurred. Thus, we suggest that overexpression of Mycprotein is toxic to a cell, unless accompanied by other,uncharactenized, transforming events.

Novel Oncogenic Cooperativity in Cell Transformation.It has been proposed that activation of c-myc in associationwith a block to apoptosis, such as that provided by consti-tutive bc/-2 expression, may represent a novel form ofoncogene cooperation in the stepwise progression of tumordevelopment (31 , 41 ). We provide an example of a non-contact-inhibited, tumonigenic cell line, L929, which hasacquired mutations in the pathways regulating c-myc geneand c-Myc protein expression as well as programmed celldeath. Furthermore, somatic cell hybridization of theseL929 cells to nontransformed rat fibroblasts functionallycomplements genetic lesions inhibiting the Myc-inducedapoptotic mechanism, and the resulting hybrids are capableof growth inhibition upon cell-to-cell contact. It is enticingto speculate that the mutations which abrogate the Mycnegative feedback and apoptosis pathways directly or mdi-rectly contribute to the L929 transformed phenotype. Theobservation that L929 cells have acquired not one but

644 c-myc Activation, Autoregulation, and Apoptosis

several distinct lesions affecting each of these pathwayssupports the theory of a multistep process of tumonigenesis.What is not evident is the temporal order of acquisition ofthe mutations or the causal relationship among them. Forinstance, does a cell procure an activated survival mecha-nism in response to deregulated c-Myc expression, or doesconstitutive inhibition of the apoptotic mechanism allow acell to tolerate subsequent mutations which activate thec-myc gene? Clearly, a deeper understanding of the molec-ular mechanisms of both c-myc activation and Myc-induced apoptosis is required to define the nature of thisnovel form of oncogenic cooperation.

Materials and Methods

Retroviral Vedors and Cell Lines. The pDORneor, pDoKv-myc/neor, (1 8, 1 9) and the pBabehygror (42) retroviral vec-tons were described previously. The pBabev�myc/hygrorvector was constructed by subcloning a 3.3-kilobase SpeI-EcoRl v-gag-myc fragment, derived from the avian myelo-cytom#{224}MC29 retrovirus (43), into the pBabehygror vector.

L929 is a spontaneously immortalized cell line derivedfrom adult mouse connective tissue, is tumonigenic in irra-diated mice, and is a thymidine kinase-deficient derivativeof the original clone described by Sanford et a/. (44). TheRat-i cell line is a subclone of the Fischer rat embryofibroblast line F2408 (45). HL-60 is a subclone of the orig-inal promyelocytic cell line derived by S. J. Collins (46). The�Ji2 (47) and GP+E (48) packaging cell lines were used for

the production of infectious, replication-deficient ecotropicretrovi rus.

Fibroblast and GP+E cell lines were cultured in a-mod-ified Eagle’s medium, and HL-60 cells were cultured inRPMI 1640, both supplemented with 10% FBS (GIBCO).The t2 cells were cultured in a-modified Eagle’s mediumsupplemented with 1 0% newborn calf serum (GIBCO). Allmedia were supplemented with 1 00 pg/mI kanamycin and2 pg/mI gentamicin. All cell lines tested negative for My-cop/asma infection and were routinely incubated at 37#{176}Cwith 5% CO2.

Retroviral vector DNA (1 0 pg) was transfected into theGP+E packaging cell line by the calcium phosphate pre-cipitation method (49). Retrovirus stock was harvested frompooled, drug-resistant colonies and was used to infect f i-broblast cells following the method described by Mann eta/. (47). Fibroblast and packaging cell lines containing ret-roviral vectors expressing the neomycin resistance gene(ned) were selected in 1 mg/mI G4i8 sulfate (Geneticin;GIBCO) and cells containing retroviral vectors expressingthe hygromycin resistance gene (hygro’) were selected atthe following concentrations of hygromycin B (SigmaChemical Co.): GP+E, 100 pg/mI; Rat-i , 150 pg/mI; L929,300 pg/mI.

RNase Protedion. RNA from subconfluent, proliferatingcells was prepared by the guanidmnium isothiocyanatemethod of Chirgwin et a!. (50). The RNase protection pro-cedure was conducted as described by Penn et a/. (1 8, 19).Briefly, 32P-radiolabeled antisense RNA probes were tran-scribed from linearized Bluescript KS or 5K cloning vectors(Stratagene) using T3 RNA polymerase (Stratagene). The ratc-myc exon I and v-gag-myc probes are as described (18).To generate a control probe to quantitate the RNA analyzedamong samples, a 230-base pair EcoRI-HindlIl fragment ofthe munine glyceraldehyde phosphate dehydrogenase(GAPDH) gene (a kind gift of M. Prystowsky and D. Sabath,

University of Pennsylvania, Philadelphia, PA; Ref. Si) wassubcloned into the Bluescnipt KS cloning vector. Gel-pun-fied probes were hybridized in excess to 10 pg RNA over-night in solution at 52#{176}C.Samples were digested with 40pg/mI RNase A (Sigma) and 2 pg/mI RNase T1 (Sigma) at30#{176}C,ethanol precipitated, and resuspended in loadingdye. The protected probes were denatured, resolved on8% denaturing polyacrylamide gels, and visualized byautoradiography.

Somatic Cell Hybridizations. Somatic cell hybridiza-tions were conducted as described previously (1 8), excepthybrid cells were selected for 2 to 3 weeks in mediumcontaining i mg/mi G41 8 sulfate and 300 pg/mI hygromy-cm B. The following pairs of cell lines were hybridized:Rat-i neor x L929 hygror; Rat-i v�myc/neor x L929 v-myc/hygrcf; hybridization controls: Rat-i � � Rat-i hygror;Rat-i v-myc/necf x Rat-i v�myc/hygror; L929 ned X L929hygror; L929 v�myc/neor x L929 v�myc/hygror. In addition,to control for drug selection, each parental cell line washybridized to itself.

Immunoblot Assay. Whole cell extracts were preparedfrom HL-60 and subconfluent, proliferating Rat-i and L929cells by boiling cells in sample buffer containing 1 mtvtEDTA (52). Proteins were resolved on 10% denaturing so-dium dodecyl sulfate-polyacrylamide gels as described byLaemmli (52) and electrotransferred onto nitrocellulosemembrane (Shleicher and Schull) according to the manu-facturer’s recommendations (Bio-Rad). The membrane wasblocked overnight in 10% (w/v) nonfat milk powder (Car-nation) in TBS-T [20 m� Tnis (pH 7.5)-i 37 mrvi NaCI-O.05%(v/v) Tween 20] and then washed for 1/2 h in TBS-T. Mycprotein was detected by incubating the membrane with ai :2000 dilution of polyclonal pan-Myc rabbit antiserum (akind gift of G. Evan, Imperial Cancer Research Fund, Lon-don, U.K.) in i%(w/v)nonfat milk powder in TBS-Tfonl h.The membrane was washed as before and incubated with a1 :2500 dilution of horseradish peroxidase-conjugatedswine anti-rabbit antibody (Dako) in i % milk-TBS-T for 1/2h. Following washing, the signal was visualized by en-hanced chemiluminescence (Amersham) on Kodak X-Omatfilm. Autoradiogram signal intensities were quantitated onan LKB Ultrascan XL enhanced laser densitometer.

Apoptosis and DNA Fragmentation Analysis. The fol-lowing cell lines were examined for apoptosis: Rat-i neor;Rat-i v�myc/neor; L929 neor; L929 v-mydnecf; four ran-dom clones of Rat-i ned x L929 hygror; and four randomclones of Rat-i v-myc/ned x L929 v�myc/hygror somaticcell hybrids. Quadruplicate cell cultures were seeded at 1 xiO� cells/well in 24-well culture plates in medium supple-mented with 1 0% FBS. After incubation for 2 days, mediumwas removed, and two wells of each cell line were givenmedium with 1 0% FBS; the remaining two wells were fedmedium with 0.1% FBS. Cultures were examined by mi-croscopy after 1 , 4, and 7 days for evidence of cell death.Photomicrographs were taken using Polaroid film on aNikon Diaphot-TMD inverted microscope.

To assay for DNA fragmentation, a 1 00-mm culture dishof each cell line listed above was exposed to 1 0 ml mediumcontaining 0.1% FBS. After 1, 4, and 7 days, the mediumand dead cells, if any, were collected, and the monolayerwas once again given 1 0 ml medium containing 0.1 % FBS.Cells in the harvested media were pelleted at low speed andwashed twice in chilled phosphate-buffered saline. DNAwas extracted as described by Meyaard et a!. (53). Tovisualize DNA fragmentation, samples were electropho-

Cell Growth & Differentiation 645

resed through 1 .5% agarose or 1 .0% agarose-2.0% NuSeive(FMC Corp.) gels at 70 V for 2 to 4 h, respectively. The gelswere stained with ethidium bromide and examined underan UV light source.

AcknowledgmentsWe thank G. Evan and T. Littlewood for providing the polyclonal pan-Mycantiserum, I. Kerr for the L929 cells, and M. Prystowsky and D. Sabath forproviding the mouse GAPDH cDNA used in these experiments. We extendspecial thanks to G. Wasfy for technical assistance; to i. Daksis, B. Gallie, C.Guidos, R. Lu, W. Marhin, C. McDowell, R. Phillips, and i. Squire for helpfuldiscussions and critical review of the manuscript; as well as S. Thompson for

secretarial assistance.

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