The RAS oncogene induces genomic instability in thyroid ... - Nature

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Oncogene (2000) 19, 3948 ± 3954 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

SHORT REPORT

The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway Harold I Saavedra2,3, Jerey A Knauf1,3, Jill M Shirokawa1, Jianwei Wang1, Bin Ouyang3, Rosella Elisei1, Peter J Stambrook2 and James A Fagin*,1,2 1

Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Cincinnati, Ohio, OH 45267, USA; Department of Anatomy, Neurobiology and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio, OH 45267, USA

2

Activating mutations of RAS are thought to be early events in the evolution of thyroid follicular neoplasms. We used a doxycycline-inducible expression system to explore the acute eects of H-RASV12 on genomic stability in thyroid PCCL3 cells. At 2 ± 3 days (®rst or second cell cycle) there was a signi®cant increase in the frequency of micronucleation. Treatment of cells with YVAD-CHO inhibited RAS-induced apoptosis, but had no eect on micronucleation. The eects of H-RASV12 were mediated by activation of MAPK, as treatment with PD98059 at concentrations veri®ed to selectively inhibit MEK1 reduced the frequency of prevalence of cells with micronuclei. In addition, doxycycline-inducible expression of a constitutively active MEK1, but not of a mutant RAC1, mimicked the eects of H-RASV12. The eects of H-RASV12 on genome destabilization were apparent even though the sequence of p53 in PCCL3 cells was con®rmed to be wild-type. Acute activation of H-RASV12 evoked a proportional increase in both CREST negative and CREST positive micronuclei, indicating that both clastogenic and aneugenic eects were involved. H-RASV12 and activated MEK1 also induced centrosome ampli®cation, and chromosome misalignment. Evidence that acute expression of constitutively activated RAS destabilizes the genome of PCCL3 cells is consistent with a mode of tumor initiation in which this oncogene promotes phenotypic progression by predisposing to large scale genomic abnormalities. Oncogene (2000) 19, 3948 ± 3954. Keywords: genomic instability; Ras; thyroid cancer; MAP kinase

Follicular thyroid cells give rise to neoplasms that vary in their cellular characteristics, histopathological appearance and biological behavior. Progress has been made in identifying oncogenic events responsible for initiating tumor clones that ultimately may evolve into these distinct phenotypes. Perhaps the best established of these genotype/phenotype associations is the *Correspondence: JA Fagin, Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, The Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, Ohio, OH 45267, USA 3 The ®rst two authors contributed equally to this work Received 27 December 1999; revised 26 May 2000; accepted 1 June 2000

involvement of rearrangements of the RET tyrosine kinase oncogene as an early event in the development of papillary thyroid carcinomas (Grieco et al., 1990; Jhiang et al., 1996), particularly after exposure to ionizing radiation (Nikiforov et al., 1997). Autonomously functioning thyroid adenomas are associated with activating mutations of the thyrotropin (TSH) receptor (Parma et al., 1993) or Gsa (Lyons et al., 1990). These benign tumors rarely undergo malignant transformation (Porcellini et al., 1997). By contrast, point mutations of RAS are seen in both follicular adenomas and carcinomas (Namba et al., 1990; Suarez et al., 1988). Mechanisms whereby constitutive activation of particular signal transduction pathways may predispose cells to undergo malignant transformation, whereas others do not, have not been resolved. Thyroid cells represent an interesting model in which to explore these questions, as mutations of signaling intermediates along dierent transduction cascades have been implicated in tumor initiation. The rate of spontaneous mutations acquired during the natural life span of a cell is low (Oller et al., 1989; Thacker, 1985). This suggests that one of the early genetic disruptions involved in tumor development may confer cells with a `mutator' phenotype (Loeb, 1997), hence predisposing to the accumulation of additional abnormalities. In many cells RAS promotes unregulated cell division, and cooperates with other oncogenes to induce cell transformation (Fusco et al., 1987). Replacement of a normal H-RAS1 gene by an activated mutant H-RAS by homologous recombination in rat1 ®broblasts is not in itself sucient to induce transformation, but rather requires secondary events such as gene ampli®cation events, including ampli®cation of the mutant RAS allele (Finney and Bishop, 1993). Induction of expression of the human H-RAS oncogene in p53-null NIH3T3 cells leads to premature entry of cells into S phase, increased permissivity for gene ampli®cation, and generation of aberrant chromosomes within a single cell cycle (Denko et al., 1994; Wani et al., 1994). Overexpression of oncogenic RAS has also been shown to produce chromosome aberrations in rat mammary carcinoma cells (Ichikawa et al., 1990), rat prostatic tumor cells (Ichikawa et al., 1991) and in a human colon carcinoma cell line (de Vries et al., 1993). Although RAS mutations are found in both thyroid follicular adenomas and carcinomas, they are more prevalent in the latter (Esapa et al., 1999). Moreover, follicular carcinomas have a higher rate of aneuploidy

Ras induces genomic instability in thyroid cells HI Saavedra et al

and chromosomal aberrations (Tung et al., 1997; Ward et al., 1998). These observations suggest that activating mutations of RAS may initiate the formation of thyroid follicular neoplasms and promote their progression by decreasing genomic stability. To explore this possibility we have modi®ed the rat thyroid cell line PCCL3 to undergo conditional expression of a variety of signaling intermediates involved in thyroid cell transformation. PCCL3 cells are derived from rat thyroid follicular cells and are dependent on TSH for growth. They also retain dierentiated properties as they trap iodide, express thyroglobulin, thyroid peroxidase, and the TSH receptor in a TSH-dependent manner. PCCL3 cells constitutively expressing the tetracycline-dependent transcription factor rTTA were used as hosts for secondary stable transfections with cDNAs coding for activated mutants of various signaling proteins cloned downstream of a tetO-sensitive promoter, using a previously described method (Gossen et al., 1995). Figure 1 illustrates conditional expression of H-RASV12, MEK1, RET/PTC1, RET/PTC3, and RAC1V12 in the respective clonal lines, as demonstrated by very low or undetectable basal expression of the appropriate gene product, and a 4 ± 20-fold induction after incubation with the tetracycline analog doxycycline.

Figure 1 Doxycycline-inducible expression of constitutively activated oncogene products in PCCL3 thyroid cells. Western blots of extracts from the indicated cell lines incubated with or without doxycycline for 24 hours. (a) rTTA (vector-transfected controls), Ras-25 or Ras-39 (clones with inducible expression of H-RAS (val 12)) probed with an anti-RAS IgG. (b) rTTA or MEK1-55 and MEK1-65 (with inducible expression of constitutively active MEK1 (Glu-217/Glu221)) probed with an antiMEK1 IgG. (c) Rac-88 and Rac-90 (inducible expression of a myc epitope-tagged, constitutively active RAC1 (val12)) probed with an anti-myc IgG. (d) rTTA, PTC1-21, or PTC3-31 clones (inducible expression of either RET/PTC1 or RET/PTC-3) probed with an anti-RET IgG. The membranes were incubated with the indicated IgG and later with their corresponding speciesspeci®c horseradish peroxidase-conjugated secondary IgG and visualized using the ECL non-radioactive detection system. All antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA)

We ®rst explored the eects of acute overexpression of oncogenic RAS on genomic stability of PCCL3 cells, as detected by formation of micronuclei. Micronuclei are small nuclear-like structures, which contain chromosomes or chromosome fragments that form during mitosis as a result of chromosome missegregation. This approach has been used successfully to measure the extent of chromosomal loss resulting from DNA-damaging agents such as ionizing radiation, or toxins that interfere with the proper functioning of the mitotic spindle (Miller and Nusse, 1993; Muller et al., 1996). To validate ¯ow cytometry as an approach to assay for micronuclei in PCCL3 cells, an rTTAexpressing clone was exposed to g-irradiation. As predicted, the percentage of cells with micronuclei increased in a dose-dependent manner (Figure 2a). Thyroid cells are remarkably resistant to radiationinduced apoptosis in vitro (Yang et al., 1997). Indeed 48 h after irradiation with 5 ± 20 Gy there was no signi®cant increase in apoptosis in PCCL3 cells. Addition of doxycycline signi®cantly increased the percentage of cells with micronuclei in Ras-25 and Ras-39 cells, but not in rTTA controls (Figure 2b). The higher prevalence of micronuclei in Ras-25 and Ras-39 cells in the absence of doxycycline likely represents either clonal variation, or leakiness of the promoter (i.e. low-level expression of H-RASV12 in the absence of the antibiotic). In support of the latter, we observed that Ras-39 cells have higher levels of expression of the mutant protein than the Ras-25 clone (Figure 1), as well as higher levels of micronuclei in unstimulated conditions (Figure 2b). In both cases, unstimulated levels were higher than in rTTA vector control cells (Figures 1 and 2). Micronuclei cannot be distinguished from apoptotic bodies by ¯ow cytometry. This is an important consideration, as acute activation of expression of mutant RAS also triggers programmed cell death in PCCL3 cells, albeit with dierent kinetics than that of micronuclei formation (Shirokawa et al., 1999). To con®rm the increase in micronuclei, we also determined the acute eects of RAS in micronuclei formation by cytochemistry. This allows apoptotic bodies to be distinguished from micronuclei by their appearance after PI staining by ¯uorescence microscopy, as the former are associated with cells whose nuclei are fragmented, whereas true micronuclei are seen within cells that have an intact nucleus. Induction of RAS expression resulted in an increase in micronuclei of a similar magnitude to that seen by ¯ow cytometry (Table 1). Addition of YVAD-CHO, a cell permeable inhibitor of caspase-1 and -4, suppressed RAS-induced apoptosis by 50%, but had no eect on RAS-induced micronuclei formation (Figure 2c). Moreover, the cytochemical analysis demonstrated that the level of micronuclei was maximal 4 days after the addition of doxycycline, whereas the number of cells with apoptotic bodies did not increase signi®cantly until day 8 (Figure 2d). To determine the predominant signaling pathways used by mutant RAS to induce genomic instability, expression of the oncogene was induced in the presence of PD98059, which inhibits activation of MAPK. PD98059 was used at the minimal concentration veri®ed to block RAS-induced MAPK phosphorylation in PCCL3 cells, as determined by Western blotting with a phospho-speci®c antibody (not shown). Treat-

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Figure 2 Formation of micronuclei after exposure to g-irradiation or acute activation of RAS in PCCL3 thyroid cells. (a) Cells were harvested 48 h after exposure to the indicated dose of g-irradiation and the number of micronuclei determined by ¯ow cytometry. The micronucleus assay was performed as described (Nusse et al., 1994). Bars represent the mean+s.e.mean of three experiments performed in triplicate. *P5261077 versus untreated cells. (b) Cells were harvested 4 days after addition of doxycycline to induce expression of RAS and the number of micronuclei determined by ¯ow cytometry. Bars represent the mean+s.e.mean of ®ve experiments performed in triplicate. *P51.961077 versus cells grown in the absence of doxycycline. (c) The caspase 1 and 4 inhibitor YVAD inhibits RAS-induced apoptosis, but not micronuclei formation. Number of apoptotic cells and micronuclei in rTTA and Ras-25 cells incubated with or without the caspase inhibitor YVAD for 8 days in the presence of doxycycline. YVAD was replenished every 12 h. Bars represent the mean+s.e.mean of two experiments performed in duplicate. *P50.05 versus cells treated without YVAD. (d) Time course of RAS-induced micronuclei and apoptosis in Ras-25 cells. Cells were incubated either with or without doxycycline for the indicated times. Bars represent the mean+s.e.mean of two experiments performed in duplicate. *P50.002 versus untreated cells. The number of micronuclei and apoptotic cells were counted as described in Table 1 Table 1 Eects of ras on formation of micronuclei: distinction from apoptotic bodies by visual counting Cell line rTTA-Dox rTTA+Dox Ras-25-Dox Ras-25+Dox

% Micronuclei

% Apoptosis

5.93+1.03 6.82+1.00 7.38+0.73 26.50+1.08

1.29+0.27 1.55+0.33 1.19+0.17 3.21+0.35

Detached and attached cells were combined, washed with ice-cold PBS, spotted onto a microscopic slide, and ®xed in 50/50 methanol/ acetone. Cells were washed with PBS containing 0.1% Triton-X100 and then with PBS containing RNAse A. Cover slips were mounted in vectorshield anti-fade solution containing SYTOX and propidium iodide. The number of nuclei, micronuclei (de®ned as small propidium iodide and SYTOX-staining structures not associated with fragmented nuclei), and apoptotic cells (cells with fragmented nuclei) per ®eld was determined. For each condition more than 2000 cells were counted. Numbers shown are the mean+s.e.mean of two experiments scored by two operators blinded to the identity of the cell line and treatment

ment with PD98059 blunted the RAS-induced increase in micronuclei (Figure 3a). By contrast, PD98059 had Oncogene

no eect on micronuclei levels in rTTA cells. The involvement of MAPK in mediating genomic destabilization by RAS was de®ned further in PCCL3-derived cell lines modi®ed to conditionally express a constitutively active mutant of MEK1 (Figure 1). Induction of expression of MEK1-Glu-217/Glu221 in MEK1-55 and MEK1-65 resulted in an increase in micronuclei (Figure 3b), thus con®rming the involvement of MAPK in this process. MEK1 is a dual speci®city MAP kinase kinase, that is activated by various serine/ threonine protein kinases, especially members of the Raf family (Alessi et al., 1994; Dent et al., 1992). After activation, MAP kinases translocate to their site of action at several subcellular locations, including the nucleus (Chen et al., 1992). MAP kinases are wellestablished regulators of events in the G0?G1?S stages of the cell cycle (Boulikas, 1995; Meloche, 1995). MAP kinase has also been shown to function as an eector for Mos in meiosis (Kosako et al., 1994), and is a component of the spindle checkpoint in Xenopus egg extracts (Bitangcol et al., 1998; Minshull et al.,


1994). There is compelling evidence that MAP kinases may also play an important role in somatic cell mitosis (Shapiro et al., 1998; Zecevic et al., 1998). Activated MAP kinase localizes to kinetochores in early and midmitosis, in asters during all stages of mitosis, and in the midbody in late anaphase (Zecevic et al., 1998). It associates with the motor protein CENP-E and phosphorylates it in vitro. CENP-E localizes to kinetochores during prometaphase and regulates attachment of chromosomes to microtubules. MAP kinase also phosphorylates proteins containing the 3F3/2 phosphoantigen, which recognizes an epitope that disappears with kinetochore attachment to the spindles (Shapiro et al., 1998). It is reasonable to propose that the gross mitotic abnormalities associated with constitutive activation of RASV12 and MEK1 in thyroid cells may have resulted from interference with these interactions of MAP kinase. Evidence for a temporal sequence of localization of activated MAP

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Figure 4 Eects of forskolin on micronuclei formation in PCCL3 cells. Ras-25 and rTTA cells were grown in TSH-free medium for 4 days in the absence or presence of forskolin and with or without doxycycline. Cells were then harvested and the number of micronuclei determined by ¯ow cytometry. Bars represent the mean+s.e.mean of two experiments performed in triplicate. *P5261076 versus cells grown in the absence of doxycycline

Figure 3 RAS-induced micronuclei formation requires activation of MAP kinase. (a) rTTA, Ras-25 and Ras-39 cells were incubated with or without 35 mM PD98059 in the absence or presence of doxycycline for 4 days and then assayed for micronuclei by ¯ow cytometry. Bars represent the mean+ s.e.mean of at least three experiments performed in triplicate. *P51.961077 versus untreated cells. **P5461075 versus cells treated with doxycycline, but without PD98059. (b) Eects of induction of expression of a constitutively active MEK1 mutant on micronuclei. MEK1-55, MEK-65, and rTTA cells were incubated for 4 days in the absence or presence of doxycycline and then assayed for micronuclei by ¯ow cytometry. Bars represent the mean+s.e.mean of at least three experiments performed in triplicate. *P51610714 versus untreated cells. **P51.561073 versus untreated cells. (c) Eects of acute expression of other oncogene products on micronuclei formation in PCCL3 thyroid cells. Cells were harvested 4 days after addition of doxycycline to induce expression of either RAC1V12, RET/ PTC1, or RET/PTC3 and the number of micronuclei determined by ¯ow cytometry. Bars represent the mean+s.e.mean of at least three experiments performed in triplicate. *P510714 versus cells grown in the absence of doxycycline

Table 2 Acute expression of oncogenic ras induces a proportional increase in aneugenic and clastogenic chromosomal events

Cell line rTTA-Dox rTTA+Dox Ras-25-Dox Ras-25+Dox

Number of micronuclei scored

CREST (+)

% CREST (+)

245 241 244 178

144 136 136 103

58.8 56.4 55.7 57.9

Ras-25 and rTTA cells were incubated in the absence or presence of Dox for 4 days, ®xed in a 1 : 1 mixture of methanol/acetone, and stained with propidium iodide and for CREST immunoreactivity. The CREST antibody recognizes the centromeric proteins CENP-A, -B and -C, and can be used to determine if the micronuclei contain a kinetochore. Five optical sections of cells containing micronuclei were analysed on a Biorad confocal microscope (Kalman mode, 1006objective). The micronuclei not staining for CREST in any of the optical sections were considered CREST negative, where as those staining positive for CREST in any of the optical sections were considered positive Oncogene


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kinase in dierent nuclear compartments during mitosis (Shapiro et al., 1998; Zecevic et al., 1998) suggests that phosphorylation-dephosphorylation steps are needed for orderly progression, a step that may be disrupted when MAP kinase activation is constitutive. Besides signaling through Raf-MEK1-MAP kinase, RAS also activates the c-Jun amino-terminal kinase (JNK) and p38 MAP kinase in PCCL3 cells (Shirokawa et al., 1999). Mitotic spindle disruption of mammalian cells with nocodazole is associated with activation of p38 MAP kinase, but not JNK or MAP kinase (Takenaka et al., 1998). However, acute

expression of RAC1V12, which signals constitutively through SEK-JNK or SEK-p38MAPK in PCCL3 cells, did not induce micronucleation suggesting that activation of these kinases through alternative eectors is not sucient to trigger this process (Figure 3c). The chimeric gene products generated by the rearrangement of the RET gene (RET/PTC1 or RET/PTC3) are believed to be involved in initiation of papillary thyroid carcinomas (Grieco et al., 1990; Jhiang et al., 1996; Powell et al., 1998; Viglietto et al., 1995). However, neither doxycyclineinduced expression of RET/PTC1 nor RET/PTC3

Figure 5 Mitotic defects resulting from acute expression of activated RAS. (a) A normal mitotic Ras-25 cell incubated without doxycycline. Centrosomes (in yellow) were visualized with rabbit anti-g-tubulin, mitotic spindles (in red) were visualized with mouse anti-b-tubulin, and chromosomes (in blue) were visualized with DAPI. (b) A representative mitotic cell with a misaligned chromosome found in Ras-25 cells incubated with doxycycline. Arrow indicates the misaligned chromosome. (c) A representative mitotic cell with centrosome ampli®cation found in Ras-25 cells incubated with doxycycline. The four centrosomes associated with mitotic spindles are indicated by the arrows. (d) Representative Ras-25 cell with a mitotic bridge. (e) Quanti®cation of mitotic defects in the indicated cell lines incubated with or without doxycycline for 3 days. Bars represent the mean+s.e.mean of three experiments. *P50.045 versus rTTA cells grown in the presence of doxycycline Oncogene


increased the frequency of micronucleus formation (Figure 3c). Cells with inactivating mutations of the p53 gene are predisposed to gene ampli®cation, aneuploidy and other large scale chromosomal aberrations, including those triggered in other cell types by mutations of RAS (Agapova et al., 1999). To determine the status of p53 in the PCCL3 cell line, p53 cDNA was obtained by RT ± PCR with overlapping sets of intron-spanning primers, and the products used as templates for DNA sequencing. The expressed p53 products were found to have normal sequence. Furthermore, abundance of p53 was increased by treatment with DNA damaging agents, with concomitant increase of p53-dependent transcriptional activity as assessed by transient transfection with a p53-responsive reporter construct and increased abundance of p21WAF1 and Bax (Shirokawa et al., 1999). Thus, the eects of constitutive activation MAPK on genome destabilization were not dependent on concomitant loss-of-function of the p53 tumor suppressor. It is possible that accumulation of micronuclei arises as a consequence of increased growth rate triggered by RAS activation, rather than through eects on genomic stability. Growth of PCCL3 cells is dependent on TSH, or of agents that stimulate adenylyl cyclase activity. Although both TSH and forskolin markedly stimulated cell proliferation (data not shown), they did not have a signi®cant impact on micronucleation, indicating that this process cannot be triggered simply by stimulating cell division (Figure 4). These data are consistent with the fact that thyroid tumors with mutations of the thyrotropin (TSH) receptor (Parma et al., 1993) or Gsa (Lyons et al., 1990; O'Sullivan et al., 1991; Suarez et al., 1991) which constitutively stimulate adenyl cyclase activity, result in autonomously functioning thyroid adenomas which rarely progress to overt malignancy. Micronuclei are generally believed to form either by disruption in the mitotic spindle, leading to the loss of a whole chromosome (an aneugenic event), or by induction of double-strand DNA breaks with loss of a portion of the chromosome (a clastogenic event). Micronuclei resulting from aneugenic events can be identi®ed by staining with the anti-centromeric antibody CREST. By contrast, micronuclei resulting from clastogenic events would primarily consist of chromosome fragments, and would stain negative for CREST (although fragments that include a centromere may be CREST positive). Staining of micronuclei in RAS or MEK1-overexpressing cells with CREST revealed no change in the ratio of CREST-positive and CRESTnegative micronuclei, suggesting that oncogenic RAS induces both clastogenic and aneugenic events (Table 2). These data suggest that constitutive signaling by RAS triggers the development of chromosomal abnormalities within the ®rst few cell cycles after activation. Indeed, RAS resulted in a marked increase in misaligned chromosomes (Figure 5b,e) and centrosome ampli®cation (Figure 5c,e) 3 days after induction of expression of the oncogene. Doxycycline-induced activation of MEK1 also evoked centrosome ampli®cation and chromosome misalignment (not shown). Whereas mitotic bridges were found in a greater

proportion of nuclei of both Ras-25 (Figure 5d,e) and MEK1-55 cells (not shown) than in rTTA controls, this phenomenon was not inducible after treatment of cells with doxycycline. The increase in CREST-positive micronuclei is consistent with the higher frequency of chromosome misalignment, and may be due in part to disruption of interactions between chromosomes and microtubules by unregulated MAP kinase activation. This is in agreement with evidence that activated MAPK leads to spindle defects in p53-null NIH3T3 cells, which ultimately leads to increased ploidy (Fukasawa et al., 1995) and loss of whole chromosomes (Saavedra et al., 1999). The clastogenic eects suggest that there is also an engagement of alternative mechanisms of chromosome damage. The activity of topoisomerase II, an enzyme that generates regulated strand breaks to ensure proper condensation of chromosomes during mitosis (Daum and Gorbsky, 1998), has been shown to be modulated by RAS (Woessner et al., 1990) or ERK2 (Shapiro et al., 1999), and is postulated to be involved in mediating chromosomal breaks and recombination induced by oncogenic RAS (de Vries et al., 1993). Interestingly, topoisomerase II shares the 3F3/2 antigenic epitope, suggesting that it may be a substrate of MAP kinase, and thus subject to malfunction when this signaling pathway is compromised. Activation of oncogenic RAS in thyrocytes is associated with changes in cellular responsiveness to TSH (Francis-Lang et al., 1992). Although TSH signals primarily through activation of adenylyl cyclase (Roger et al., 1995) and phospholipase C, recent data points to the ability of TSH to transiently activate RAS (Kupperman et al., 1993; Miller et al., 1997). Adding to the complexity of this signaling network, cAMP substantially modi®es the interaction of RAS with its downstream eectors (Miller et al., 1998), and can also activate other RAS ± GEFs in a protein kinase Aindependent manner. Many of these eectors converge on MAP kinase, although the duration and intensity of activation varies considerably according to the triggering stimulus (York et al., 1998). Oncogenic events, such as RAS mutations, that disrupt this tightly regulated temporal modulation of MAP kinase have deleterious eects on mitosis, and hence may predispose to phenotypic progression.

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Acknowledgments The RASV12 and the RAC1V12-myc-tagged cDNA's were a generous gift from Dr. Kenji Fukasawa. The MEK1-Glu217/Glu221 cDNA was kindly provided by Dr. Christopher Marshall. The RET/PTC1 and RET/PTC3 cDNA's were kindly provided by Dr. S.M. Jhiang. This work was supported in part by NIH grants CA50706 and CA72597 (JA Fagin), F32CA69711 (JA Knauf), F32CA80389 (JM Shirokawa), and CA65769 (PJ Stambrook).

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