been shown to be a potent oncogene. We have devel- oped a cellular model for the study of p80 action in rat la fibroblasts. Expression of eDNA's encoding.
The activated anaplastic cellular proliferation rat la fibroblasts
lymphoma kinase increases and oncogene up-regulation
AXEL WELLMANN, VICTORIA DOSEEVA, WAYNE FUKUSH1MA, MARYALICE STETLER-STEVENSON, Laboratory
of Pathology,
National
Institutes
of Health,
BUTSCHER, AND KEVIN Bethesda,
MARK RAFFELD, GARDNER’
Maryland
20892-1500,
in PAULA USA
More than 60% of anaplastic large-cell lymphomas (Ki-1 lymphoma) are associated with a t(2;5)(p23;q35) translocation that produces an 80 kDa hyperphosphorylated chixnenc protein (p8O) derived from the fusion of the anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM). The NPM-ALK chimeric gene isan activatedtyrosine kinase that has been shown to be a potent oncogene. We have developed a cellular model for the study of p80 action in rat la fibroblasts. Expression of eDNA’s encoding NPM-ALK (p80) in rat la fibroblasts induces anchorage-independent growth in soft agar and promotes foci formation in culture. Cells expressing exogenous p80 showed significantly increased proliferation characterized by accelerated cell cycle entry into S-phase. Consistent with increased Go/G1 to S-phase transition, there is also marked up-regulation of cydlln A and cydlin Dl expression. In addition, p80 transformed cells showed elevated expression of several immediate early genes involved in cellular proliferation, including fos, jun, and c-myc. DNA binding analysis of nuclear extracts prepared from p80 transformed cells reveal marked up-regulation of AP-l DNA binding activity. Functional AP-l-spedflc transfection assays also show up-regulation of AP-l-dependent transcriptional activation. These finding demonstrate that p80 transformed rat la fibroblast can be a highly useful model system for the molecular and biochemical characterization of the mechanisms of action of this interesting new oncogene.-Wellmann, A., Doseeva, V., Butscher, W., Raffeld, M., Fukushima, P., Stetler-Stevenson, M., Gardner, K. The activated anaplastic lymphoma kinase increases cellular proliferation and oncogene up-regulation in rat la fibroblasts. FASEBJ. 11, 965972 (1997)
such proteins is often associated with malignant transformation and many other alterationsin normal cellular behavior (1-3). Although many types and classes of both protein tyrosine kinases and receptor PTKs have been identified and studied, litde has been determined about the functional mechanisms through which alterationsin their downstream signaling pathways, targets, and events ultimately lead to the evolution of the oncogenic phenotype. Anaplastic large-cell lymphoma (Ki-1 lymphoma) is one of the more commonly occurring forms of T cell lymphoma in humans. Lymphomas of this type are generally considered tumors of activated T cells, and at the time of diagnosis frequently involve both nodal and extranodal sites including skin, lung, bone, and submucosal tissues (4). The majority of these tumors (>60%) are associated with a t(2;5) (p23;q35) chromosomal translocation that produces an active 80 kDa gene product (p80), which is the result of a chimeric fusion of the nuclear protein nucleophosmin (NPM) with a novel tyrosine kinase identified as the anaplastic lymph oma kinase (ALK) (5). The ALK is a previously unrecognized member of the insulin receptor protein tyrosine kinase family (5). Deregulation of its catalytic activity by fusion to NPM underlies its oncogenicity, and it has been clearly shown that p80 can act alone to cause the transformation of immortalized rodent fibroblasts (6, 7). Recent studies indicate that the major role of the NPM portion of the chimera is to provide a dimerization motif that facilitates activation of the tyrosine kinase domains via autophosphorylation (6, 7). Evidence for a direct physical association between the p80 and phosphotyrosine binding Src homology adaptor proteins Grb2, Shc, and IRS-i suggests that p80 may function
Key Words: membrane
‘Correspondence: National Institutes of Health, Bldg. 10, Rm. 2N212, Bethesda, MD 20892-1500, USA. 2Abbreviations: ALK, anaplastic lymphoma kinase; CAT,
ABSTRACT
plasmid antibody
vector PTK
gene
expression
cytosolic
(PTKs)2 are widely known to play a major role as mediators of the signal transduction events that control cellular growth, proliferation, and differentiation. Deregulated expression of PROTEIN
TYROSINE
KINASES
0892-6638/97/0011 -0965/$02 .25 © FASEB
chloramphenicol acetyl transferase; EMSA, electrophoretic mobility shift assay; FACS, fluorescent-activated cell sorting; FCS, fetal calf serum; NPM, nucleophosmin; PMSF, phenylmethylsulfonyl fluoride; PTK, protein tyrosine kinase; RTPCR, reverse transcription-polymerase chain reaction; TRE, TPA-responsive
element.
965
through Ras-dependent pathways (6,7). Although a cogent hypothetical scheme explaining the general mode of p80 action is beginning to emerge from these most recent findings, precise delineation of the cellular and molecular pathways induced by p80 during the evolution of the transformed phenotype remains undefined. To develop a better understanding of the molecular mechanism of p80 action during cellular transformation, we have constructed a cellular model for the molecular and biochemical analysis of p80-induced oncogenesis from the rat la fibroblastsystem. In this work we show that p80 can potently transform rat la fibroblasts. Consistent with their increased mitogenic activity, p80 transformed cells show up-regulated expression and function of several proto-oncogenes linked to increase cellular growth and proliferation. Possible mechanistic correlation between gene activation in p80-expressing cells and the transformed phenotype are discussed.
were selected by electroporation
AND METHODS
reading
frame
of p80/NPM-ALK
transcription-polymerase
chain
was generated reaction
by
(RT-PCR)
from RNA derived from an established anaplastic large-cell lymphoma T cell tumor line, Karpas 299, that expresses high levels of p80 due to the t(2;5) translocation (8). The forward primer used was the oligonucleotide CCC4AGCTTGGGATGGAAGATI’CGATGGA, which introduces a 5’ HIND III restriction site (shown in italics) at the amino terminus of the p80 open reading frame. The reverse primer used was the oligonucleotide ccGCCGAGGGGrCAGGGCCCAGGCrGGT, which introduces an XhoI restriction site (italics) at the carboxyl terminus of the open reading frame. The RT-PCR product was subcloned into and amplified in the pTA plasmid vector (Invitrogen, San Diego, Calif.). Two different p80 expression vector constructs were generated with the HindIH/XhoI fragment, containing p8O, expressed under control of either the CMV promoter of pCDNA 3.1 (Invitrogen) or the RSV promoter of the episomal expression vector Rep9 (Invitrogen). The forward oligonucleotide primer used to screen for p80 expression by RT-PCR was TCCCTFGGGGGCTTTGAAATAACACC. The reverse oligonucleotide primer used was CGA-GGTGCGGAGCITGCTCAG (5). The 12-O-tetradecanoylphorbol 13-acetate (TPA) -responsive element (TRE)-chloramphenicol acetyl transferase (CAT) and icE-CAT reporter plasmids have been previously described
(9, 10). Tissue culture
and
gene expression/transformation
assays
Rat la fibroblasts (gift from C. V. Dang, Johns Hopkins University School of Medicine) were grown and maintained in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL, Gaithersburg, Md.) with 10% fetal calf serum (FCS), and 100 U/mI of penicillin/streptomycin at 37#{176}C in 5% CO. Cell viability was determined by the trypan blue exclusion assay. Cells were transfected with control pRep9 or pRep9-p80 expression vectors by lipofection (lipofectamine; Gibco-BRL), and selected
966
Vol. 11
according to with 500 tg/ml
October
1997
the
agar Serum
pooled.
Transfection
described
(9).
performed as destudies were per-
assays were deprivation
as described phenicol
(9). Percent was
phoimager
quantitated
(Sunnyvale,
conversion on
of ‘4C-labeled
a Molecular
chloram-
Dynamics
phos-
Calif.).
Preparation of cytosolic membrane
and
nuclear
extracts
rat la cells (approximately 8X106-3X107) were washed three times in ice-cold phosphate-buffered saline. The cells were quickly scraped into 3.5 ml of phosphate buffer and pelleted by centrifugation at 1000 gat 4#{176}C. The cells were then resuspended in five volumes of buffer A (10 mM KC1, 1.5 mM Adherent
MgCl2, 4 mM f3-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 .tg/ml leupeptin, 10 mM Hepes, pH7.5) and transferred to a 1.5 ml Eppendorf tube and al-
to stand for 10 mm at 4#{176}C. Next the cells were collected by centnfugation at 1000 gfor 10 mm at 4#{176}C. The supernatant was removed and the cells were resuspended in two volumes of buffer A. The cells were homogenized with 16 strokes of a lowed
for 1.5 ml Eppendorf tubes) driven at 0.75 HP power drill. Nuclei were collected by centrifugation at 1000 gfor 10 mm at 4#{176}C and the supernatant was removed. The membrane components of the supernatant where then pelleted by centrifugation at 300,000
Plasmid vectors
reverse
and soft
and
out as previously
scribed previously (11). formed in 0.2% serum for 24 h or as indicated. CAT activity in extracts from transiently transfected cells was determined
manufacturers specifications, of G418. Drug-resistant colonies
pestle
(fitted
rpm by a hand-held
1500
The open
wk of growth
was carried
Focus formation
Teflon
MATERIALS
after 2
g for 30 mm. The cytosolic supernatant was removed and retained; the membrane pellet was resuspended in three volumes of 10 mM Tris, 50 mM NaC1, 0.5 mM PMSF. Nuclear extracts were prepared from the isolated nuclei as previously described (12). Electrophoretic
mobility
shift
assays
Electrophoretic
mobility
shift
analysis
(EMSA)
was carried
out
with 5’ end-labeled duplex oligonucleotides as previously described (13). Oligonucleotides used in this study are as follows: APi-consensus (GALV-TRE) 5’-GATCCCGAGAAATAGATGAGTCAACAGCGA-3’; kappa B consensus (HIV-ic.B) 5’-GATCGAGCTrGCTACAAGGGAC1TrCCGCTGGGG-3’. Antibodies Antibodies to p80 have been described previosuly (14). Antibodies to cyclin A, cyclin Dl, and c-jun were obtained from Santa Cruz (Santa Cruz, Calif.). Antibodies top65 were a generous gift from Uli Siebenlist (NIAID, Bethesda, Md.). Antibodies to the fos protein were raised against the M peptide common to all fos family members and was a generous gift from Michael ladorolla (NINDS, Bethesda, Md.). Anti-junD
antibodies were previously described (13). Western blot analyses were performed as already described (15). Cross-reactive proteins were visualized by (Amersham, Arlington Heights,
facturer’s
specifications.
Cell
analysis
cycle
the Ill.)
ECL
detection
according
system
to the manu-
Cellular
synchronization was performed by serum starvation plus 0.2% FCS. At the times indicated, cells were processed for DNA synthesis and content analysis as previously described (16). DNA content was measured using a FACScan flow cytometer (Beckton-Dickinson, San Jose, Calif.), calibrated with Autocomp beads (Beckton-Dickinson), and stained with chicken red blood cells (RBC). Data acquisition in media
The FASEB Journal
WELLMANN
ET AL.
of p80 in all three cellular compartments is consistent with the pattern previously seen by immunofluorescent staining and subcellular fractionation of established tumor cell lines containing the t(2;5) translocation (7, 17). Disregulated or uncontrolled growth is a primary phenotype of transformed cells. This feature is often characterized by the ability of the cells to 1) show growth patterns that are independent of cell density, 2) grow independently of adherence or anchorage to the underlying substrate, and 3) have reduced serum dependence for growth and survivability. Density-independent growth is commonly manifested in cell culture by the ability to form 3-dimensional foci during growth on monolayers. Compared to control cells, which are transfected with an empty expression vector,p8O transformed cellsshow a nearly 3000-fold increased ability to form foci (Fig. 2A, B). Moreover, p80-expressing cellsalso show increased anchorageindependent growth when grown in soft agar (Fig. 2C).
A
B
‘so C,
cytosol
nuclei
A
membrane
a -p80 Figure blasts.
RNA
1. Stable expression A) RT-PCR analysis from control (empty
confirms
of p80
(NPM-ALK)
(see Materials
Empty Vector
p80
in rat la fibro-
and Methods)
of total
vector) and p80 transformed cells of a 177 nt product that is also present
the presence
in the Karpas 299 tumor cell line derived from a patient with anaplastic large-cell lymphoma, shown here as a positive control (8). The bottom panel shows equivalent loading and quality of the total RNA derived from the control and p80-expressing cells. B) Western blot analysis of cellular fractions from control and p80 transfected cells shows the presence of an 80 kDa cross-reactive band present exclusively in the p8O transformed cells. Approximately 30 tg of cytosolic,
B
nuclear, and membrane protein isolated from control and p80 transformed cells were separated by SDS page and subject to immunoblot analysis with dicates position of the 80
was carried munocytometry
anti-p80
antibodies.
Arrowhead
kDa cross-reactive
out using CeIIFIT
software
in-
bands.
(Becton-Dickinson
Im-
C
Empty
Vector
p80
Systems).
RESULTS Stable expression fibroblast
of p8O transforms
rat la
Stable expression of p80 in rat la fibroblastwas confirmed by RT-PCR analysis (Fig. l#{192}). Western blot analysis of nuclear, membrane, and cytosolic fractions prepared from mock-transfected and p8Oexpressing cells reveals the presence of the p80-encoded 80 kDa polypeptide exclusively in the p8O transfected cells (Fig. 1B). The ubiquitous expression
REGULATED
SIGNAL
TRANSDUCTION
BY p80
Figure 2. p80 expression potently transforms rat Ia fibroblasts. Rat la fibroblasts were stably transfected with expression vectors encoding p80 (pRep-p80) or mock transfected with an empty vector. A) Foci formation assay of mock- (left) and p8Otransfected rat la fibroblasts. B) Morphology of representative transformed foci produced by p80-expressing cells compared to mock-transfected cells. C) Representative colonies produced by the p80 transformed cells after growth in soft agar
(see Material and days of growth.
Methods).
Images
were captured
after
14
967
24-hour Starvation
24- hour Starvation
+
16 hours with serum
Control
p80-expressing cells show marked the S-phase of the cell cycle.
acceleration
into
p80 Transformed cells show up-regulated early expression of the G1 cydlins and increased levels of the mitogenic proto-oncogenes jun, fos, and c-myc pRep-p80
Figure
3. p80
transformed
cells
show
accelerated
cell
cycle
entry. Control and p80 transformed cells were synchronized by serum deprivation in 0.2% serum for 24 h. The cells were then placed in media containing 10% serum for 16 h. Cells at zero and 16 h after serum deprivation were analyzed for DNA content and nucleic acid incorporation by FACScan analysis (Materials and Methods). Cell count (y-axis), DNA content (xaxis), and nucleic acid incorporation (z-axis) are depicted in 3-dimensional format.
p80 Transformed the cell cycle
cells show accelerated
entry
into
The disregulated growth pattern of the p80 transformed cells suggests that these cells are likely to show significant changes in cell cycle progression. To test this possibility, p80-expressing and mock-transfected cells were arrested and synchronized by serum starvation for 24 h and then returned to 10% serum for 16 h. Cell progression was measured by BrdU and propidium iodide incorporation, followed by fluorescent-activated cell sorting (FACS) analysis. As shown in Fig. 3 (left), both control cells and p80-expressing cells (pRep-p80) show complete arrest after 24 h serum starvation. However, after serum release, the
To investigate the general mechanisms that underlie the abilityof p80 to propel rat la fibroblasts through the cell cycle, we surveyed the status of several cellular components important for cell cycle progression and serum-dependent induction of mitogenesis. Parental and p80 transformed cells were synchronized by serum starvation as in Fig. 3 and then pulsed for 16 h with 10% serum. Cell samples were harvested before and after the 16 h serum pulse and nuclear extracts were prepared (Fig. 4). The G1 cyclins cyclin Dl and cyclin A play a major role in promoting cell cycle progression to S-phase during proliferative responses. The expressions of both cyclins are periodic. Under the mitogenic stimulation provided by serum growth factors, cyclin Dl expression peaks during early G1 and then declines as the cells enter S-phase and approach G2/M (18). After mitogenic stimulation, cydin A expression does not peak until middle to late G1 (18). Immunoblot analysis of nuclear extracts from p80 transformed cells that have been synchronized by serum deprivation show a marked premature increase in cyclin A before the addition of serum (Fig. 4A). This increase continues to persist 16 h after the addition of serum, when cyclin A levels begins to normally accrue in the parental cells (late G1). Similarly, the levels of cyclin Dl are significantly elevated in serum-starved p8O transformed cells compared to parental control (Fig. 4A). Unlike cyclin A, but similar to that of the parental cells, the levels of cyclin Dl in the p80 transformed cells decreases signifi-
B
A &
,
0,
.O. -3
.“
MrxlO
&
.O
-c-fos
A
-cyclrA
46-
a-fos/Frci
_____________
46-
ct-CYCIIflD1
.,
-
MrxlO3
“
68c-cyclin
c/ \O
Fra 1,2 29-
4FosB
46-
i#{225}
a-jun
.-cyclinD1
29-
a-c-myc 68 464. p80 transformed cells show increased levels ofjun, fos, and c-myc. Western blot analysis of nuclear extracts from serumstarved parental or p80 transformed cells before and after a 16 h pulse with serum. Extracts were analyzed for the presence of cyclin A and cyclin Dl (A) and fos,jun, and c-myc (B) by immunoblot analysis.
Figure
968
Vol. 11
October
1997
The FASEB Journal
WELLMANN
ET AL.
cantly by late G1. This difference suggests that, at least in the case of cyclin Dl, p80 expression may result in a general increase in the synthesis of cyclin Dl in early G1 rather than a decrease in its net degradation throughout the cell cycle. Thus, pSO expression results in both increased and unscheduled production of cyclin A and cyclin Dl in rat la fibroblasts. The AP-l family of proteins plays a large role in mediating cellular proliferative responses. They are a dimeric family of transcription factors that form either Jun Jun homodimeric complexes composed of Jun family members (c-Jun, JunD, and jun B) or FosJun heterodimeric complexes composed of fos family members (c-Fos, Fral, Fra2, and FosB). Immunoblot analysis of rat la nuclear extracts with a pan-fos antibody specific for the M peptide common to all fos family members (19) shows up-regulation of FosB in serum-starved cellsexpressing p80 (Fig.4B). In addition, the serum-inducible expression of c-fos and the fos related antigens 1 and 2 (Fra 1,2) isalso dramatically increased in the p8O transformed cells. Coincident with the elevated levelsof fosfamilymembers, p80-expressing cells also show a large increase in the amount of jun proteins (c-jun and junD) in both quiescent and serum-induced cells (Fig. 4B, center panel). The role of c-myc as a potent oncogene involved in several hematopoietic malignancies is well known (20). Its immediate early expression is a common component of the proliferative responses of a wide variety of cells and can potently transform rat la fibroblasts. Immunoblot analysis shows up-regulated expression of c-myc in quiescent p80-expressing cells and less pronounced differences after serum induction (Fig. 4B, lower panel). #{192}Y-i DNA binding activity and transactivation selectivelyup-regulated in p8O transformed fibroblasts
are rat la
The marked up-regulation of the AP-l factors in p80 transformed cells can be correlated with functionally increased DNA binding activityin nuclear extracts prepared from control and p80 transfected fibroblasts (Fig. 5A). EMSA of DNA binding to a consensus AP-l binding site (TRE) in nuclear extracts from p80expressing cells shows marked up-regulation of serum-induced TRE DNA binding activity (Fig. 5A). Although there is no detectable AP-1-specific DNA binding activity in quiescent or serum-induced control cells, DNA binding activity in serum-stimulated p8O transformed cells is increased more than 50-fold. The nonspecific DNA binding activity seen in the control and uninduced nuclear extracts of p80-expressing cellsisdue to an abundant low-affinity activity contributed by the Ku nuclear antigen (21). This complex is not blocked by AP-1-specificantibodies, and only the specific serum-induced AP-l DNA/protein complex is supershifted by anti-jun antibodies
REGULATED
SIGNAL
TRANSDUCTION
BY p80
(Fig. 5A, right panel). Consistent with the elevated AP-1 expression and DNA binding activity, rat la cotransfection experiments using control and p8O expression vectors in combination with a TRE reporter CAT driven by three tandem copies of a AP-1 binding site show a p80-dependent increase in transactivation from the TRE promoter (Fig.5B). AP-1 activityis elevated through both transcriptionaland posttranslationalmechanisms regulated by immediate early events (22). In contrast, the NF-xB p65/pSO factors are members of a ubiquitous family of inducible transcription factors whose activityis posttranslationally controlled mainly by modulation of the subcellular localization of p65/p50 complex in the cytoplasm before cellularstimulation (23). In comparison to AP-l, the NF-kappa B-specificDNA binding activity is only modestly up-regulated in quiescent and serum-induced cells (Fig. 5C). Accordingly, xB-dependent transcriptionalactivityis only slightly increased by cotransfections experiments with p80 expression vectors (Fig.5D).
DISCUSSION In this work we have confirmed that p8O is a potent transforming agent when expressed in rat la fibroblasts. Cellular and biochemical characterizationof p80 transformed cells reveals that p80-expressing cells show increased cell cycle entry associated with disregulated expression of specific cyclins and immediate early genes, including c-myc and the fos/jun AP-l family of proto-oncogenes. Functional analysis confirms specific up-regulation of the transcriptional activity of the AP-l. The evolution of the transformed phenotype during oncogenesis occurs through a complex set of intersectingand overlapping cellularevents involving changes in both mitogenic regulation and cellcycle progression. Nearly every aspect of these cellular processes is controlled by coordinated inputs from multiple signal transduction pathways in the cell.The p80 transformed rat la cell system will serve as an effectivemodel system through which mechanisms of oncogenesis can be studied by direct comparison among cells that differ genetically by a single gene product. Use of thismodel system has allowed us to demonstrate p80-dependent up-regulation of both cyclinA and cyclin Dl, two key modulators of cycle progression in eukaryotic cells. Cyclin Dl expression and function are common targets of oncogenic events in a varietyof hematopoietic malignancies (24, 25). Since cyclinA expression istightlyregulated and anchorage dependent (26),the p80-induced up-regulation of cyclinA expression in the p80 transformed cells is consistent with their anchorage-independent phenotype.
969
A
B Nuclear
Extracts
30 25
4-a-jun
immune AP-1
i
15
#{149}. AP-1 0 nonspecific
20
complex
‘
-+
10
I-nonspecIc 5
0 TRE DNA -4
CMV-vector CMV-p80 RSV-p80
D
C Nuclear Extracts
p65/pso
p50/pso
4a-p65/p65/DNA knmune
-ox
0
kB DNA
-4. RSV-vector RSV-p80
Figure 5. p80 Transformed cells show differential up-regulation of AP-i and NF-kappa B-dependent DNA binding activity and transcriptional transactivation. A) Left panel: EMSA analysis of nuclear extracts from synchronized populations of control and p80-expressing rat la fibroblasts (prepared as described in Fig. 3), using a 32P-labeled oligonucleotide encoding a TRE consensus sequence (see Materials and Methods). Short arrow indicates position of specific AP-1/DNA complexes. The faster migrating
complex (unmarked) represents a ubiquitous nonspecific binding activity common to the nuclear extracts of various cell types (20). Right panel: Anti-jun supershift experiment demonstrating inhibition and altered mobility of the specific AP-1 protein/ DNA complexes. B) Rat la fibroblasts were cotransfected with 5 p.g of either an empty expression vector (CMVvector, pCDNA3.i) or expression vectors encoding p80 (CMV-p80, pCDNA-p80; and RSV-p80, Rep9-p80) in the presence of 2.5 pg of a TRE-CAT expression vector (see Materials and Methods). Cells were harvested 72 h after transfection and assayed for CAT activity. The data represent the average and standard error of duplicate experiments. C) Left panel: EMSA analysis of nuclear extracts from synchronized populations of control and p80-expressing rat la fibroblasts using a 32P-labeled oligonucleotide encoding a consensus iB binding site (see Materials and Methods). Short arrow indicates position of p65/p5O and pSO/pSO dimers. Right panel: Antibody supershift analysis of specific p65 containing DNA/protein complexes indicating specific NF-icB complexes with DNA. D) Cells were transfected with 5 tg of empty Rep9 vector or the Rep-p80 plasmid in the presence of 5 j.tg of a cB-CAT reporter plasmid construct and processed as described in Fig. 5B. The data are the average and standard error of transfections
performed
970
Vol.11
in triplicate.
October
1997
The
FASEB Journal
WELLMANN
ET AL.
The cyclinA and cyclinDl promoters both contain enhancer elements that can be induced by transcription factorcomplexes containing AP-l and the AP-lrelatedATF-2. There isalsoa potentialc-myc binding sitein the cyclinDl promoter (27).The up-regulated expression of fos/jun and c-myc therefore provides a potentialmechanistic link between the altered signal transduction pathways in p80 transformed cellsand the control of cellcycle progression. Recent elegant structure-function analysis of p80 has been carried out through point mutation and deletion studies of the tyrosine kinase domain (6, 7). These studieshave shown thatcertain interactionsof p80 with effector molecules that up-regulate Ras function have no influence on the transforming abilityof the cells(6, 7). Thus, certain signal transduction pathways augmented by p80 are likely to affect other cellularfunctions that are independent of the transformed state.Itwillbe important in the future to examine how such mutants are altered in their specific targeting for the signal transduction pathways and downstream transcriptional effectors of p80 action. The observations that pSO transformed cellsare readily arrested by serum withdrawal and can be easily synchronized by serum deprivation suggest that other mitogen-dependent pathways parallel to or upstream of p80 strongly modulate p80-dependent signaling. Unlike c-myc transformed rat la fibroblasts, which readily undergo accelerated apoptosis when serum deprived (28), p80 transformed cells do not (data not shown). These observations suggest that p80 and c-myc may use significantly different pathways to effect transformation. Such functional differences will serve as a useful comparative system to decipher oncogene specificpathways of tumorigenesis. Future molecular and biochemical characterization of p80 transformed cells will lead to a fuller elaboration of the mechanism of p8O transformation in rat la cellsand willalso shed new light on the general and tissue-specific mechanisms of cellular growth, cell cycle regulation, and tumorigenesis. jj
A.W. was supported by a grant from the Dr. Mildred Stiftung, Deutsche Krebshilfe, Bonn, Germany.
2. 3. 4.
5.
Ullrich, A., and Schiessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203-212 de Ran, R. and van ‘t Veer, M. B. (1993) Clinical features ofCD3O (Ri-I) positive anaplastic large-cell lymphoma (ALCL). Review of the literature. Neth. j Med. 43, 277-284 Morris, S. W., Kirstein, M. N., Valentine, M. B., Dittmer, K. C., Shapiro, D. N., Saitman, D. L., and Look, A. T. (1994) Fusion of
SIGNAL
8.
9.
10.
11.
TRANSDUCTION
BY p80
(1997) Role of the nucleophosmin (Npm) portion of the nonHodgkins lymphoma-associated Npm-anaplastic lymphoma kinase fusion protein in ocogenesis. Mol. Cell. Biol., 17, 23122325 Fischer, P., Nacheva, E., Mason, D. Y., Shemngton, P. D., Hoyle, C., Hayhoe, F. G., and Karpas, A. (1988) A Ri-I (CD3O) -positive human cell line (Karpas 299) established from a high-grade non-Hodgkin’s lymphoma, show a 2;5 translocation and rearrangement of the T-cell receptor beta-chain gene. Blood 72, 23440 Farina, A. R., Davis-Smyth, T., Gardner, K., and Levens, D. (1993) An early response of an AP1-junD complex during T-cell activation. J. Biol. Chem. 268, 26466-26475 Schmidt, A., Hennighausen, L., and Siebenlist, U. (1990) Inducible nuclear factor binding to the kappa B elements of the human immunodeficiency virus enhancer in T cells can be blocked by cyclosponn A in a signal-dependent manner. J. Virol. 64, 4037-4041 Hoang, A. T., Lutterbach, B., Lewis, B. C., Yano, T., Chou, T. Y., Barrett,J. F., Raffeld, M., Hann, S. R., and Dang, C. V. (1995) A
link between increased transforming activity of lymphoma-derived MYC mutant alleles, their defective regulation by plO7, and altered phosphorylation of the c-Myc transactivation domain. Mol. Cell. Biol. 15, 4031-4042 12.
13.
14.
15.
16.
17.
18. 19.
20.
Hunter, T. (1996) Tyrosine phosphorylation: past, present and future. Biochem. Soc. Trans. 24, 307-327 Hunter, T., Lindberg, R. A., Middlemas, D. S., Tracy, S., and van der Geer, P. (1992) Receptor protein tyrosine kinases and phosphatases. Cold Spring Harb. Symp. Quant. Biol.57, 25-41
REGULATED
7.
Scheel
REFERENCES 1.
6.
a kinase gene, ALK, to a nucleolar protein gene, NPM, in nonHodgkin’s lymphoma [published erratum appears in Science (1995) Vol. 267, 316-317). Science263,1281-1284 Fujimoto,J., Shiota, M., Iwahara, T., Seki, N., Satoh, H., Mori, S., and Yamamoto, T. (1996) Characterization of the transforming activity of p80, a hyperphosphozylated protein in a Ki-l lymphoma cell line with chromosomal translocation t(2;5). Proc. Nail. Acad. Sri. USA 93, 4181-4186 Bischof, D., Pulford, K., Mason, D. Y., and Morris, S. W.
21.
22.
23.
24.
Powers, C., Krutzsch, H., and Gardner, K. (1996) Modulation of JunD/AP1 DNA binding activity by AF-l. J. Biol. Chem. 271, 30089-30095 Gardner, K., Moore, T. C., Davis-Smyth, T., Krutzsch, H., and Levens, D. (1994) Purification and characterization of a multicomponent AP-l.junD complex from T cells. Dependence on a separate cellular factor for enhanced DNA binding activity. J. Biol. C/zen. 269, 32963-32971 Shiota, M., Fujimoto,J., Takenaga, M., Satoh, H., Ichinohasama, R., Abe, M., Nakano, M., Yamamoto, T., and Mori, S. (1994) Diagnosis of t(2;5)(p23;q35) -associated Ki-l lymphoma with immunohistochemistry. Blood 84, 3648-3652 Bennett, V. and Davis,J. (1981) Erythrocyte ankyrin: immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain. Proc. Nail. Acad. Sci. USA 78, 7550-7554 Seynaeve, C. M., Stetler-Stevenson, M., Sebers, S., Kaur, C., Sausville, E. A., and Worland, P.J. (1993) Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res. 53, 2081-2086 Mathew, P., Sanger, W. G., Weisenburger, D. D., Valentine, M., Valentine, V., Pickering, D., Higgins, C., Hess, M., Cui, X. L., Srivastava, D. K., and Morris, S. W. (1997) Detection of the t(25) (p23-q35) and NPM-ALK Fusion in Non-hodgkins lymphoma by 2-color fluorescence in-situ hybridization. Blood 89, 16781685 Sherr, C.J. (1996) Cancer cell cycles. Science 274, 1672-1677 Curran, T., Van Beveren, C., and Verma, I. M. (1985) Viral and cellular fos proteins are complexed with a 39,000-dalton cellular protein. Mol. Cell Biol. 5, 167-172 Kato, G.J. and Dang, C. V. (1992) Function of the c-Myc oncoprotein. FASEBJ 6. 3065-3072 Quinn,J. P. and Farina, A. R. (1991) Autoimmune antigen Ku is enriched on oligonucleotide columns distinct from those containing the octamer binding protein DNA consensus sequence. .FEBS Leit. 286, 225-228 Siebenlist, U., Franzoso, G., and Brown, K. (1994) Structure, regulation and function of NF-kappa B. Annu. Rev. Cell Biol. 10,405455 Karin, M. (1995) The regulation of AP-1 activity by mitogenactivated protein kinases. J. Biol. Chem. 270, 16483-
16486 Lukas,J., Bartkova,J., and Bartek,J. (1996) Convergence of mitogenic signalling cascades from diverse classes of receptors at
971
25.
26.
972
the cyclin D-cyclin-dependent kinase-pRb-controlled Cl checkpoint. Mol. Cell Biol. 16, 69 17-6925 Bartkova,J., Lukas,J., Strauss, M., and Bartek,J. (1995) Cyclin Dl oncoprotein aberrantly accumulates in malignancies of diverse histogenesis. Oncogene 10, 775-778 Shulze, A., Zerfass-Thome, K., Berges,J., Middendorp, S.,Jansen-Durr, P., and Henglein, B. (1996) Anchorage-dependent transcription of the cyclin A gene. Mol. Cell. Biol. 16, 46324638
Vol. 11
October
1997
27.
Herber, B., Truss, M., Beato, M., and Muller, regulatory
elements
in the human
R. (1994) cyclin Dl promoter.
Inducible Oncogene
9, 2105-2107 28.
Evan, G. I., Wylie, A H., Gilbert, C. S., Uttlewood, T. D., Land, H., Brooks, M., Waters, C. M., Penn, L Z., and Hancock. D. C. (1992) Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119-128
The FASEB Journal
Recei ved for publication May 20, 1997. Accepted for publication Ju ne 20, 1997.
WELLMANN
FT AL
