oncogene is able to dedifferentiate a rat thyroid epithelial cell
line in culture. N o t a di M a s s ~ o
SANTORO, R O S ~ X ~ N A
MELmLO, MICHELE G ~ c o , VECCHIO,
dal Corrisp. G. Salvatore.
ABSTRACT. - - We have recendy cloned a new oncogene from human papillary thyroid carcinomas, which we have designated PTC. This oncogene consists of a chimeric gene, the 5'-end of which corresponds to a gene of unknown function, designated H4, while the 3' portion corresponds to the tyrosine kinase domain of the RET proto-oncogene. We constructed a retroviral vector containing the PTC oncogene under the transcriptional control of the Moloney LTR, and then produced the corresponding defective retrovirus by transfecting the rett,oviral vector onto Psi-2 cells. We subsequently used this retrovirus to infect a differentiated rat thyroid cell line (PC-CI-3) which is dependent on TSH for growth. Upon infection with the PTC virus the PC-C1-3 cells no longer depended on TSH for growth. Furthermore, the infected cells lost completely their differentiated functions, i.e. thyroglobulin and TSH-receptor genes expression and iodide uptake. However, they retained their non malignant phenotype in that they were not able to grow in semisolid media nor in the athymic mice. The introduction in the PC-C1-3 cell line of both the PTC and the activated viral Ha-ras oncogenes resulted in the appearance of a completely undifferentiated and malignant phenotype. These results demonstrate that: a) The PTC oncogene, which plays an important role in human thyroid carcinogenesis, is also able to induce the complete dedifferentiation of rat thyroid cells in vitro; b) Two cytoplasmic oncogenes (Ha-ras and PTC) cooperate in the induction of a fully malignant phenotype of a thyroid differentiated cell line.
KEY w o m s : Thyroid; Oncogene; Tyrosine-kinase.
R~XSSUNTO. - - L'oncogene PTC induce la perdita del fenotipo dtfferenziato di cellule epiteali tiroidee di ratto in coltura. I] nostro laboratorio ha recentemente clonato un nuovo oncogene da carcinomi tiroidei papillari umani: tale oncogene ~ stato denominato PTC. Tale sequenza trasformante 6 una chimera costituita dal dominio tirosino-chinasi del proto-oncogene RET e da una porzione amino-terminale di un gene in via di caratterizzazione che ahbiamo denominato H4. Abbiamo costruito un vettore retrovirale contenente l'oncogene PTC sotto il controUo trascrizionale della **Long Terminal Repeat~ del virus della Leucemia di Moloney (MoLTR) e abbiamo successivamente generato il corrispettivo retrovirus difettivo trasfettando tale vettore nelle cellule competenti per il packaging(Psi-2). I1 retrovirus cosi ottenuto ~ stato utitizzato per infettare una linea cellulare epiteliale differenziata di tiroide di ratto, le PC-C1-3, la quale necessita di ormone tireotropo (TSH) per la crescita. In seguito all'infezione tali cellule sono diventate indipendenti dal TSH e hanno perso completamente i loro ,*markers~ di differenziazione, quali sintesi e secrezione di tireoglobulina, espressione del recettore per II TSH e captazione degli ioduri. Le cellule tuttavia hanno mantenuto il loro fenotipo non m.aligno in quanto non si sono dimostrate capaci di crescere in terreno semisolido nd di indurre tumori se inoculate nel topo atimico. Invece l'introduzione contemporanea di PTC e dell'oncogene virale Ha-ras helle cellule PC-C1-3 ha indotto un fenotipo completamente trasformato e indifferenziato. Questi risultati dirnostrano che: a) l'oncogene PTC, che svolge un ruolo importante neUa cancerogenesi tiroidea umana, 6 anche in grado di indurre una completa dedifferenziazione in cellule tiroidee in vitro; b) due oncogeni citoplasmatici (Ha-ras e PTC) sono in grado di cooperare nella induzione di un fenotipo completamente maligno in questa linea tiroidea differenziata.
(*) Nella seduta dell'8 febbraio 1992.
M. SANTORO ET AL.
Tumorigenesis is thought to be a multistep process (Hunter, 1991); in fact the exponential relationship of cancer incidence to age suggests that multiple events are required to lead a cell to a fully malignant phenotype. However, it is only recently that some of the molecular events that underlie the initiation and the progression of human tumors have been identified (Bishop, 1991). These include activating mutations of cellular proto-oncogenes as well as inactivation of tumor-suppressor genes (Hunter, 1991). Also in vitro transformation is known to be a mulfiphasic process as demonstrated in fibroblast primary culture. Immortalizing genes, i.e. those capable of rescuing primary cultures from senescence, must operate in concert with transforming oncogenes, such as ras or the middle T gene of Polyoma virus, to obtain transformation of primary fibroblasts (Land et al., 1983). We have reported the activation of a novel oncogene (designated PTC) in about 20% of human thyroid papillary carcinomas (Fusco et al., 1987b ; Bongarzone et al., 1989; Grieco et al., 1990). PTC is a chimeric gene; in fact it is the product of the fusion of the tyrosine-kinase encoding domain of the proto-RET gene (Takahashi and Cooper, 1987) to the 5'-terminal region of a still uncharacterized gene that we have named H4 (Grieco et al., 1990). An inversion of the long arm of chromosome 10 (inv10q11.2-q21) is responsible for the activation of P T C (Jenkins et al., 1990; Pierotti et al., 1992). So far the activation of the RET oncogene has been found in vivo only in thyroid carcinomas (Ishizaka et al., 1988; Koda, 1988) and it is restricted to the papillary histotype (Santoro et al., 1992). In our laboratory a well characterized rat thyroid epithelial cell line, the PC-C1-3 cell line, is available. This cell line retains in vitro the typical markers of thyroid differentiation, i.e. thyroglobulin (Tg) synthesis and secretion, iodide uptake and dependence on thyrotropin (TSH) for growth. Thus PC-C1-3 is a suitable cell system with which to identify the steps necessary to achieve the fully malignant phenotype of thyroid cells in vitro. Since thyroid follicular cells represent in vivo a specific target for the activation of the PTC oncogene, the PC-CI-3, because of its thyroid origin, is also an ideal cell system to investigate the effect of P T C on thyroid transformation and differentiation. Here, we demonstrate that the PTC oncogene, alone, induces a block in the expression of the thyroid differentiated functions without causing the appearance of typical transformation markers. A highly malignant phenotype was achieved, instead, by the cooperation of P T C with the activated viral v-Ha-ras oncogene. MATERIALS AND METHODS
Construction of the PTC virus.
To obtain a retroviral vector able to express the activated P T C oncogene, the fulllength cDNA of P T C has been cloned in the EcoRI site of the plasmid vector pMV7 (Maddon et al., 1986). The obtained recombinant plasmid was transfected in the Psi-2 cell line (Mann et al., 1983).
THE PTC O N C O G E N E
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Cells and Transfection. Psi-2 is an NIH/3T3 murine fibroblast cell line (Mann et aL, 1983). It was grown in DMEM medium supplemented with 10% Calf serum (Flow Laboratories). The transfection was performed according the calcium-phosphate transfection protocol (Graham and Van der Eb, 1973). After 36 hours the cells were trypsinized, expanded and the antibiotic geneticin (G418) was added to the medium at a concentration of 400 micrograms/ml. The PC-C1-3 cell line is a thyroid epithelial cell line derived from eighteen-month old Fischer rats (Fusco et al., 1987a). The PC/Ha-ras cells have been previously described (Fusco et al., 1987a). The normal and infected cell lines were cultured in modified F12 medium supplemented with 5% calf serum (Flow Laboratories) and six growth factors (thyrotropin, hydrocortisone, insulin, transferrin, somatostatin and glycil-histidyl-lysine, SIGMA Chemical Corporation ).
Infection and assay of the transformed state. PC-C1-3 and PC/Ha-ras cell lines were infected with the PTC recombinant retrovirus essentially as described (Fusco et al., 1981). Briefly, viral stocks were prepared by adding 10 ml of flesh DMEM/10% CS to a near confluent monolayer of the Psi-2/PTC cell clone. After 24 hrs, the medium was removed and filtered through a 0.45 micron filter (MiUipore). For infection, 2 • 105 cells were incubated with 2 ml of viral supernatant and three days later they were reseeded into flesh medium containing 400 micrograms/ml of G418. For the assay of the transformed state, soft agar colony assays were performed according to a previously described technique (Fusco et al., 1981). The tumorigenicity of the transfected and infected cell lines was tested by injecting 2 • 106 cells into athymic mice. The animals were palpated at the inoculation site twice a week. Assay of the differentiated phenotype. Iodide uptake assays were performed as described (Colletta et al., 1983). RNA was purified from cultured cells by a modification of the guanidine thiocyanate method (Santoro et al., 1990). The assay of rat thyroglobulin (Tg), thyrotropin receptor (TSH-R) and thyroid peroxydase (TPO) genes transcripts were performed according to the Northern-blot procedure (Sambrook et al., 1989). The probes used for the Northerns were: a) a 1.3 kilobase EcoRI fragment specific for the tyrosine-kinase domain of the RET oncogene (Grieco et al., 1990); b) a 680 bp EcoRI-PstI fragment corresponding to the 3'-end of the rat thyroglobulin gene (Di Lauro et al., 1985); c) the purified insert from clone T8AFB which represents residues -54 to 2780 of the nucleotide sequence of the rat thyrotropin receptor gene, a generous gift of Dr. Leonard D. Kohn (Akamizu et al., 1990); d) a 0.7 kb EcoRI-SacI fragment corresponding to the 5'-coding region of the rat thyroid peroxydase gene, a generous gift of Dr. R. Di Lauro. Labelling of the probes was performed with the Amersham random priming oligolabelling system kit according to a previously published procedure (Sambrook et al., 1989).
M. SANTORO ET AL.
RESULTS AND DISCUSSION
Construction of the PTC virus and infection of the PC-CI-3. In an attempt to obtain a PTC virus, the cDNA of the PTC oncogene (Grieco et al., 1990) was cloned into the retroviral vector pMV7 (Maddon et aL, 1986). pMV7 contains two directly repeated Moloney murine sarcoma virus long terminal repeats (LTR) that flank a single EcoRI cloning site in which the PTC cDNA has been subcloned. In addition, pMV7 contains the herpes virus thymidine kinase promoter (tk) fused to the coding region of the bacterial neomycin phosphotransferase gene (neo), a dominant selectable marker. To obtain a recombinant retrovirus able to encode PTC sequences, the resulting recombinant plasmid (pMV7-PTC) was transfected into the cell line Psi-2 (Mann et al., 1983) according to the standard procedure of DNA-mediated gene transfer (Graham and Van der Eb, 1973). The Psi-2 is an NIH/3T3 cell line containing a defective murine ecotropic provirus; consequently it is incapable of encapsidating its endogenous viral RNA, but it can provide all obligate trans viral functions. After 36 hrs the transfected cells were selected by the addition of the neomycin analogue G418 (geneticin) to the culture media and several colonies were isolated. All the G418-resistant cell clones demonstrated a high expression of the PTC oncogene (data not shown). The expression of PTC resulted ;m a full malignant conversion of the Psi-2 cells. In fact, the Psi-2/PTC exhibited a transformed morphology with many cells looking refractile and spindleshaped (data not shown) and their injection into nude-mice resulted in the rapid (about 10 days) appearance of fibrosarcomas. The introduction of pMV7-PTC into the Psi-2 cell line resulted in the production of replication-defective recombinant retroviral stocks encoding the PTC oncogene that were free of helper virus. These pure viral stocks were then used to introduce efficiently PTC sequences into rodent cells. Supernatants from many G418-resistant Psi-2 cell colonies were tested for the production of the PTC virus by infecting NIH/3T3 murine fibroblasts with various dilutions of the filtered media and determining the numbers of transformed and G418-resistant colonies. A cell line designated Psi-2/PTC producing about 104 ffu/ml was selected for further studies. The PC-C1-3 cell line was infected with the PTC virus produced by the Psi-2/PTC cell line as described under <
Morphology, growth properties, differentiated and transformedphenotype of the PC/PTC cells. The PTC expressing cells did not show dramatic morphological changes with respect to the normal PC-C1-3 cells. They retained an epithelial morphology but showed spindles and a significant increase in refractility (fig. 1). The PC-C1-3 parental cell line requires the addition of six growth factors for the growth. We investigated whether the expression of the PTC oncogene modified this
M o r p h o l o g 3 / o f P C - C I - 3 b e f o r e a n d after infection with the PTC virus. M a g n i f i c a t i o n • 150 ( u p p e r ) ; x 3 0 0 (lower).
O Z O r t":l :Z t"/'l Va
requirement by studying the growth rate in medium with or without the six growth factors. The expression of the PTC oncogene alleviated the transfected cells from this requirement (fig. 2). No significant differences were observed between different PTC expressing cell clones.
2 | | |
Z~PC O PC A PC 9 PC
-6H + 6H PTC - 6H PTC + 6H
W4 O 3
96 120 144 168 192 216 240 HOURS
Fig. 2. - Growthcurves of the PC-C1-3cells and the PC/PTC cells in the presenceor the absence of the six growth factors.
The cell lines infected by the PTC virus were assayed for tumorigenicity in nude mice by injecting 2 x 106 cells subcutaneously and thereafter monitoring at regular intervals the appearance of tumors. Representative results are shown in table I. None of the PC/PTC cell clones was found to be tumorigenic. Also, the soft agar colony assay was negative.
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Interestingly also a human thyroid papillary carcinoma cell line that harbors an activated P T C oncogene is not tumorigenic in the nude-mice assay (Ishizaka et al., 1990). TABLE I.
-- Analysis of the neoplastic markers of the PTC infected PC-CI-3 cells.
The differentiated thyroid phenotype was followed by monitoring the transcription of the thyroglobulin (Tg) (fig. 3), thyrotropin-receptor (TSH-R) (not shown) and thyroid peroxydase (TPO) genes (not shown) by Northern blot RNA hybridization. All the PCA'YFC cell lines lost, almost completely, the expression of these differentiated functions. We also tested the ability of the infected cells to concentrate radioactive iodide (a property unique to thyroid cells in vivo and in vitro). As shown in table II, also this differentiated property was lost in the PCfI~C ceils. TABLE II. -- IodMe uptake by PC/PTC cell lines.
Counts per minute of 1251incorporated,expressedas percentageof the total 125I added to the medium.
Cooperation between the P T C oncogene and the Viral Transforming Harvey ras oncogene.
The ras oncogenes are frequently activated in human thyroid tumors (Lemoine et al., 1989; Suarez et al., 1990). Therefore the effect of the cooperation between P T C and the vHa-ras oncogene was investigated in the PC-CI-3 cell line. To this aim we infected with the P T C virus, the PC/Ha-ras (PC-C1-3 cells infected with the Harvey routine sarcoma virus, Fusco et al., 1987a). Several infected clones were isolated by the G418 selection and three of them, were tested for the acquisition of the transformed phenotype. No significant differences were observed between clones deriving from the same viral infection.
M. SANTORO ET AL.
Fig. 3. - Analysis of the expression of the differentiated functions in the PC/PTC cell lines. Northern blot hybridization of PC/PTC cell lines. 10 micrograms of total RNA for each cell line was size fractionated on a denaturing formaldehyde agarosegel, blotted onto Nylon filters Hybond-N (Amersham) and probed with a Tgspecific cDNA probe. Lane PC: uninfected PC-CI-3 cells. Lanes i, 2, 3 and 4: different PC/PTC cell lines.The 28S and the 18S ribosomal RNAswere used as molecularweight markers. Actinwas used as control for uniform RNA loading.
The cells were very refractile and spindle-shaped and, in contrast to the untransfected PC-C1-3, they did not form colonies, a feature that is typical of the normal thyroid cells (fig. 4). Since both the PC/PTC and the PC/Ha-ras cells did not express the thyroid differentiated functions (dependence on the six growth factors, iodide uptake and expression of the Tg, T S H - R and T P O genes) we evaluated the phenotypic markers of neoplastic transformation of the PC/Ha-ras/PTC ceils. The cooperation of PTC with the v-Ha-ras oncogene was able to induce the malignant phenotype in the PC-C1-3 cells (table I). These cells, in fact, induced tumors with a very short latency period (7-10 days) when injected into nude mice. The tumors were very aggressive: they reached as much as 3-4 cm in diameter and resulted in the animals' death.
THE PTC O N C O G E N E
IS A B L E T O D E D I F F E R E N T L ~ T E
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Fig. 4. Morphologyof the PC/Ha-ras/PTC cells. Magnification -
In conclusion PTC-virus infection of PC-C1-3 cells resulted in two main effects. The first concerns the cell growth: the integration and expression of the P T C oncogene rendered the cells independent of the six growth factors for growth. A similar growth pattern has been observed, for example, after the infection with retroviruses harboring ras oncogenes (Fusco et al., 1987a). Conversely, the PC-C1-3 cells infected with the Polyoma murine leukaemia virus (PyMLV) carrying the middle T gene of the Polyoma virus (Berlingieri et al., 1988) and the PC-C1-3 transfected with the Adenovirus E 1A gene (Berlingieri et al., manuscript in preparation) were still partially dependent on TSH. The second effect exerted by the P T C oncogene was on the expression of the thyroid differentiation markers: PTC was able to block their expression. Therefore, the effect of PTC on thyroid differentiation was the same as that exerted by other transforming oncogenes (Fusco et al., 1981, 1987a). Finally, we investigated the cooperation of PTC with the Ha-ras transforming viral gene. Interestingly, the complementation with v-Ha-ras induced a highly malignant phenotype. The molecular mechanisms underlying these complementations are under investigation particularly with respect to the possible involvement of growth factor induction as already described in the cooperation between v-myc and the activated human c-ras-Ha (Land et al., 1983). In any case it is interesting to observe that this is one of the few cases of cooperation between two genes belonging to the same complementation group (Land et al., 1983). In conclusion, the model of multistep epithelial carcinogenesis decribed herein is a promising tool for the study and understanding of the mechanisms of action of PTC and the processes of thyroid carcinogenesis.
M. SANTORO ET AL.
ACKNOWLEDGEMENTS The authors are grateful to Prof. Gaetano Salvatore for the continuous and enthusiastic encouragement received by him throughout the course of this work. This work was supported by the Progetto Finalizzato
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