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1 Department of Pathology and New York University Cancer Institute, New York University School of Medicine, New York, New York; 2 Molecular Pathology and Molecular Oncology Program, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain; and 3 Center for Experimental Research and Medical Studies, University of Turin, Turin, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is important to note that Ras has been previously shown to induce the expression of several cell cycle regulators, including p15INK4b (4) , and it is important to determine the ability of Rgr to produce those effects. In addition, it has been previously shown that there is Ras activation and inactivation of p15INK4b in some tumors (5) . This seemingly contradictory observation is interpreted as if the increase in p15INK4b expression produced by Ras is a defense mechanism of the cell to block the transforming effects of Ras. Therefore, it is also of interest to investigate whether Rgr would cooperate with p15INK4b inactivation as well.
Recently, we have shown that at least part of the transforming activity of Rgr is a consequence of its overexpression, which in turn is due to the elimination of translational regulatory elements in the 5' leader region of Rgr mRNA. This overexpression resulted in subsequent Ras activation (6) . The human orthologue of the rabbit Rgr gene, hrgr, has been recently isolated from lymphoid malignant cell lines that express several hrgr mutant transcripts (7) . Similar to the activated rabbit truncated form (Rgr oncogene), the human mutant transcripts found in T-cell lines and some T-cell lymphomas are truncated forms of the hrgr gene (7) .
This report summarizes the analysis of Rgr transgenic mouse lines in an effort to study the in vivo oncogenic potential of Rgr. In these experiments, Rgr has demonstrated the ability to induce pathological alterations, including tumors, in tissues in which it is expressed. Some of the neoplasic alterations were substantially increased in a p15INK4b-defective background, indicating cooperation between these two genetic alterations in some tumor types for Rgr-induced tumorigenesis. More importantly, the expression of Rgr in T cells was able to cause the formation of lymphomas with a high penetrance, consistent with the alteration found in human lymphomas and confirming the important role of this oncogene in this type of tumor.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression of oncogenic Rgr driven by the majority of the promoters listed above was analyzed in vitro before obtaining the transgenic mice. pNM11 and pCX-RGR vectors were transfected into NIH3T3 cells by the calcium phosphate precipitation method (11) , and the expression of Rgr was tested using a focus formation assay and Northern blot. The expression of RGR in pmCC10-RGR vector was confirmed by transfecting H441 lung cells by the calcium phosphate precipitation method and performing a Northern blot. In addition, the luciferase reporter plasmids (p15INK4b promoter) used in this work were described previously (4) .
For hybridizations, digested DNA or RNA was separated on agarose gels and transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH). DNA probes were labeled with [32P]dCTP (3,000 Ci/mmol; Dupont-New England Nuclear, Boston, MA) using the Random Prime DNA Labeling System Rediprime II (Amersham Biosciences, Piscataway, NJ) in accordance with the manufacturer’s protocol. Hybridizations were visualized by the use of a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) or by exposure to X-ray film.
Generation and Genotyping of Transgenic Mice.
Transgenic mice were generated as described previously (12
, 13) . Briefly, each of the transgene constructs was injected into the pronucleus of fertilized eggs from female donors and subsequently transferred to pseudopregnant mice. All of the transgenic lines were produced in the inbred strain FVB/N (14)
. Screening of positive animals for the transgene was performed by polymerase chain reaction (PCR) of DNA extracted as described previously (15)
, using forward primers specific for each of the promoters and the common reverse primer 907–884 (5'-GTGCCTGGCTGCAGGCTCCGCAGG-3'), which is specific for the transgene. The specific primers for each of the promoters were as follows: MSV-F (5'-ACCTGAAAATGACCCTGTGC-3') for the MSV-RGR lines; mCC10-147 (5'-GGTCCTCCACTGCCTGAATA-3') for the mCC10-RGR lines; PCX-F1 (5'-CAGCCATTGCCTTTTATGGT-3') for the CMV-RGR lines; and CD4-RGR-F1 (5'-GCCCACTTTTGGGTATCAGA-3') for the CD4-RGR-Flag lines. Founder animals were confirmed by Southern blot after digestion of 20 µg of genomic DNA with SacI and hybridization with a cDNA probe containing the entire sequence of oncogenic rabbit Rgr. Screening of the offspring was performed by PCR amplification of DNA with the primers described above. The animals were maintained in accordance with National Institutes of Health and New York University institutional guidelines in a pathogen-free facility.
KO-p15INK4b mice were generously provided by M. Barbacid (Spanish National Cancer Institute, Madrid, Spain) in a 129/Sv x C57BL6 genetic background. MSV-RGR/p15+/– offspring were generated as littermates from common matings and genotyped by PCR using specific primers for the RGR transgene and both the wild-type and KO p15INK4b sequences (15) . The MSV-RGR/p15INK4b–/– line was generated by crossing MSV-RGR/p15INK4b+/– mice and then maintained by mating the double mutants, MSV-RGRp15–/–.
Cell Culture and Luciferase Assays.
NIH3T3 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% calf serum (Invitrogen), penicillin G (50 units/ml), streptomycin (50 µg/ml; Gemini Bio-Products, Woodland, CA), and 500 µg/ml fungizone (Invitrogen) and incubated in standard conditions of humidity (95%), CO2 atmosphere (5%), and temperature (37°C).
For the luciferase assays, approximately 75 ng of the reporter plasmids, 25 ng of the pRL-null (as an internal control of transfection efficiency), and 400 ng of the inducer (pNM11) or empty vector (pMEXneo) were used to cotransfect NIH3T3 cells. Transient transfections were performed by lipofection following the manufacturer’s recommendations. NIH3T3 cells were plated onto 6-well plates (NIH3T3) at a density of 100,000 cells/well, grown for 24 hours, and transfected. Forty-eight hours after transfection, cells were collected, and the luciferase assay was performed in accordance with the manufacturer’s recommendations (Dual-Luciferase Reporter Assay System; Promega, Madison, WI).
Real-Time Reverse Transcription-PCR Expression Analysis.
Total RNA was isolated from tissues and cells using Trizol (Invitrogen). To generate cDNA, 1 µg of total RNA was reverse transcribed using 0.4 µmol/L of an oligo(dT)-adapter primer and SuperScript II RNase H reverse transcriptase (Invitrogen) as described by manufacturer. To determine p15INK4b mRNA expression in thymic lymphomas and wild-type thymi, real-time reverse transcription (RT)-PCR was performed using the following primers for mouse p15INK4b: E2mp15F74, 5'-CTGCCACCCTTACCAGACCTGTGC-3'; and E2mp15R257, 5'-TCTCCAGTGGCAGCGTGCAGATAC-3'. To normalize the expression levels of p15, ß-actin expression was also determined using primers QmBactin-F (5'-TGTTACCAACTGGGACGACA-3') and QmBactin-R (5'-CTTTTCACGGTTGGCCTTAG-3'). Quantitative PCR was performed with the iCycler iQ System from Bio-Rad (Hercules, CA) using SYBR Green as DNA intercalator.
Histological and Immunohistochemical Analyses.
Tissues were fixed in 10% buffered formalin at 4°C overnight, transferred to 70% ethanol, embedded in paraffin, and sectioned at 5-µm thickness. Sections were stained with hematoxylin and eosin for histological analysis.
The immunohistochemical expression of lineage-specific markers for B cells and IgM was performed on paraffin-embedded tissue sections from MSV-RGR/KO-p15INK4b and CD4-RGR lymphomas (spleen and thymus, respectively). Antibodies used were CD45R (goat antimouse B220; clone RA3-6B2; PharMingen, San Diego, CA) and rabbit antimouse IgM (DAKO, Glostrup, Denmark), respectively. A previous heat-induced epitope retrieval step was performed in a 0.1 mol/L trisodium citrate solution for 2 minutes in a conventional pressure cooker. After incubation, immunodetection was performed with the proper biotinylated secondary immunoglobulins, followed by peroxidase-labeled streptavidin (DAKO). Diaminobenzidine was used as substrate chromogen.
Immunostaining techniques were also performed in paraffin-embedded tissue sections for the detection of p53, p21, and p16 as described above. Antibodies used were as follows: rabbit antimouse p16 (M-156; 1:35 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit antimouse p21 (C-19; 1:25 dilution; Santa Cruz Biotechnology), and rabbit antimouse p53 (CM5p; 1:100 dilution; Novocastra, Newcastle upon Tyne, United Kingdom).
Flow Cytometric Analysis.
Primary thymocytes (0.5 x 106 per point) were isolated from 6-week-old wild-type and CD4-RGR mice. To characterize the different thymocyte subpopulations as well as their activation status, CD3, CD4, CD8, CD25, CD44, and CD69 markers were detected in both groups of thymocytes. Briefly, isolated thymocytes were washed twice with PBS and incubated at 4°C for 1 hour with two different sets of antibodies from CALTAG: PE-TexasRed-antiCD8, FITC-antiCD4, PE-antiCD44, and APC-antiCD25; or FITC-antiCD3 plus PE-antiCD69. Cells were washed three times with PBS and fixed with 2% paraformaldehyde. Finally, cells were analyzed by flow cytometry in a FACscan cytometer, and data were interpreted using the CellQuest program (Becton Dickinson, San Jose, CA).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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: the Moloney murine sarcoma virus (MSV), which is expressed at significant levels in the brain (16)
, eye (16, 17, 18)
and skeletal muscle (19)
and at lower levels in the kidney, pancreas (16)
, and testis; the mCC10 promoter (mouse Clara Cell 10 KD protein), which is specific for the lung (20
, 21)
, although it also drives a low level of expression in the uterus, ovaries, and epididymus of normal adults (21, 22, 23)
; and the CMV-IE enhancer plus the chicken ß-actin promoter, which is ubiquitously expressed and starting from 4-cell stage (8)
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In the case of the MSV-RGR construct, a total of 11 transgenic lines were obtained that transmitted the transgene to their progeny. Five of these lines, with varying levels of expression in several tissues, were maintained as stable lines (L3, L21 L40, L43, and L58). Two of them, L21 and L43, were characterized and studied in more detail. Fig. 2A
summarizes the RGR expression pattern as well as the major abnormalities found in the MSV-RGR lines. More than 95% of the mice bearing the transgene developed visible lens opacity at 3–6 weeks after birth. This opacity led to cataracts with swollen disoriented lens fibers and vacuolation of the lens (Fig. 2B
i and ii). Associated with the cataracts, the presence of the oncogene elicited Harderian gland adenomas in all transgenic animals. MSV-RGR mice also exhibited inguinal hernias (Fig. 2
B iii) that affected only males (90% of L43 males and 5% of L21 males), compromising male reproductive function and leading to sterility. More importantly, fibrosarcomas in the limbs and tail were found in 10% of the transgenic mice. Tumors were composed of atypical spindle-shaped cells that had a variable mitotic activity and appeared fibroblastic, arranged in intersecting fascicles, with collagenous stroma (Fig. 2B
iv). These fibrosarcomas, which developed with a latency of 6–8 months, caused paralysis of the limbs in aged mice. These results indicated that Rgr acts as an oncogene and can produce tumors when overexpressed in transgenic mice.
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, Rgr is indeed able to induce p15INK4b expression in a fashion similar to that of Ras.
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. Indeed, the severity and incidence of the tumors induced by Rgr were substantially increased when the MSV-RGR transgenic mice were crossed with the KO-p15INK4b mouse line. In fact, all of the MSV-RGR/KO-p15INK4b mice developed fibrosarcomas in the limbs, tail, and/or ears, compared with only 10% of animals in the case of the MSV-RGR/WT-p15INK4b. Furthermore, MSV-RGR/KO-p15INK4b limb tumors were significantly larger than those found in the MSV-RGR/WT-p15INK4b mice (Fig. 4A)
. Also, the latency of the fibrosarcomas diminished substantially from 24 to 32 weeks for the MSV-RGR mice in a wild-type p15INK4b background to 3 to 6 weeks in a p15INK4b-deficient one.
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and 4B
). In addition, all of the MSV-RGR/KO-p15INK4b mice displayed lymphoid hyperplasia in the spleen (Fig. 4C)
. In 50% of these cases, this phenotype was a consequence of extramedullary hematopoiesis (foci of myeloid metaplasia in the red pulp composed of granulocytic cells, erythroblasts, and megakaryocytes in different stages of maturation), an abnormal phenotype that is also found in the p15INK4b-null mice (24)
. More importantly, the remaining enlarged spleens were associated with lymphomas, a tumor type rarely found in KO-p15INK4b mice (<1%; Ref. 24
). These tumors were predominantly well-differentiated lymphomas, composed of small- to medium-sized cleaved centrocytes that usually expressed CD45R (B220) and IgM (data not shown), whereas infiltration of the white and red pulp was variable. Thus, the concurrent presence of both genetic alterations, expression of oncogenic Rgr together with the loss of p15INK4b, has a synergistic effect in the tumoral phenotype (Fig. 4D)
.
To demonstrate the specificity of p15INK4b in cooperation with Rgr in this mouse transgenic model, we studied the expression of other tumor suppressor genes by immunohistochemistry. Specifically, we chose the following for this analysis: p16INK4a (like p15INK4b, an INK4 family member that has been found to be down-regulated in many tumor types); p21kip1 (another RB function inhibitor; in this case, a member of the CIP/KIP family); and, finally, p53, the key molecule of the other critical pathway most frequently mutated in tumorigenesis. Interestingly, none of these three genes was down-regulated in any of the seven tumors analyzed (Fig. 5)
. Moreover, we did not find overexpression of p53, suggesting that this gene is not mutated in any of the analyzed tumors. Therefore, these results appear to indicate a specific role of the constitutive lack of p15INK4b in RGR-induced tumorigenesis.
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. To further analyze in vivo the role of Rgr in lymphomagenesis, new transgenic mouse lines were generated in which Rgr expression was under the control of the murine CD4 minimal promoter (lacking the CD4 silencer region) and the CD4 enhancer (Fig. 1D)
to drive the expression in CD4- and CD8-positive T cells (both single and double positive; Ref. 10
). Among the 16 founders obtained from microinjection of the CD4-RGR-Flag transgene, three independent CD4-RGR transgenic lines (lines 19, 37, and 42), were expanded and studied. The thymocytes of the animals in all three lines exhibited Rgr expression (data not shown). As shown in Fig. 6A
, many animals (83% in L19, 38% in L37, and 68% in L42) carrying the CD4-RGR construct developed thymic lymphomas. In fact, the progression of this tumor was the cause of death in all cases. Lines 19 and 42 showed similar thymic lymphoma incidence curves in the first year of life (median latency of 22 and 20 weeks and tumor-free ratios of 6.7% and 13.2%, respectively). A weaker phenotype was displayed by line 37 (median latency of 35 weeks and tumor-free ratio at 1 year of age of 37%). The neoplasms caused complete effacement of the normal thymic architecture and frequently infiltrated adjacent soft tissues and lung (Fig. 6B)
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. To characterize the putative effects resulting from the expression of Rgr in preneoplastic thymocytes, the morphological and phenotypic features of thymocyte populations in line 42 and in the less clinically aggressive line 37 were studied. Microscopic evaluation demonstrated a normal thymic architecture and no significant differences between the transgenic mice and normal control littermates (data not shown). However, a reproducible but minimal reduction in the total number of thymocytes was observed in lines 37 and 42 (Fig. 7)
, although this decrease in the number of cells of the CD4-RGR thymi was not statistically significant. Moreover, when 6-week-old mice were used to isolate and study the frequency of different T-cell subpopulations by flow cytometric analysis, significant differences were detected between CD4-RGR and wild-type thymocytes (Fig. 7)
. These differences were more pronounced in the case of the CD4-RGR mice line with shorter latency and higher incidence of thymic lymphomas (line 42). Indeed, a significant reduction was found in the thymocyte subpopulation expressing high levels of T-cell receptor (CD3high) and the single-positive CD4 and CD8 thymocytes, together with an increase in the number of CD4/CD8 double-positive cells. No significant differences were found for the percentages of CD4/CD8 double-negative thymocytes. Higher levels of CD25-, CD44-, and CD69-positive thymocytes among CD4-RGR line 42 mice were also detected. However, only the percentages of CD25- and CD69-positive thymocytes were significantly altered in the CD4-RGR line with longer latency and lower incidence (line 37).
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Finally, because lack of p15INK4b in mice carrying a MSV-RGR construct induces a more pronounced malignant phenotype, including the onset of spleen lymphomas, we investigated whether or not p15INK4b down-regulation also plays a critical role in CD4-RGR-induced lymphomagenesis. The expression levels of p15INK4b mRNA were determined in a set of CD4-RGR thymic lymphomas by real-time RT-PCR. As shown in Fig. 8
, 50% of the tumors analyzed showed a significant down-regulation of p15INK4b expression. Also, it is interesting to note that in the other 50% of the samples, p15INK4b was overexpressed, which confirmed our in vitro results on the capacity of Rgr to induce p15INK4b expression (Fig. 3)
. These results support the functional interplay between Rgr and p15INK4b and suggest that oncogenic activation of Rgr and lack of p15INK4b expression cooperate in different tumor types.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The only known substrates for the RalGEFs are the monomeric G-proteins RalA and RalB. However, oncogenic potential has not been shown for activated Ral proteins alone, although they facilitate Ras transformation, participate in cell motility, and are required for metastatic evolution of Ras-transformed cells and for Ras-induced stimulation of cyclin D1 expression (28, 29, 30, 31, 32, 33, 34) . Interestingly, Rgr also is able to activate Ras (3) , which could explain its potency as an oncogene and its ability to transform cells and promote tumor formation in transgenic mice.
Rgr and p15INK4b Cooperate in the Development and Progression of Different Tumor Types.
Tumor development and progression are associated with multiple genetic lesions. Although it is not known which altered genes are cooperating with Rgr in tumorigenic processes, one of the candidates is the tumor suppressor gene p15INK4b. In a previous study, p15INK4b produced G1 arrest in Ras-transformed cells and decreased the tumorigenic potential of Ras and Rgr in focus formation assays in mouse fibroblasts (4)
. In addition, we have shown that, like oncogenic Ras, activation of oncogenic Rgr triggers p15INK4b expression (Fig. 3)
, which could be part of a more general antioncogenic response. Therefore, similar to oncogenic Ras, Rgr and p15INK4b interact in vitro in such a way that alteration of both genes cooperates in the malignant phenotype. These results are consistent with the fact that although Rgr is able to activate both Ral and Ras, its transforming activity is dependent on Ras and less so on Ral (3)
.
The role of p15INK4b in tumorigenesis is not well defined. Although the INK4b locus is often deleted in human tumors, its loss is concomitant with that of the INK4a/ARF locus (35) . In some cases, however, deletion of p15INK4b occurs independently of p16INK4a status (36 , 37) . More recently, it has been described that a small fraction of the KO-p15INK4b mice develop different types of tumors, mainly sarcomas (24) . The high incidence of tumors observed in the double-mutant MSV-Rgr/KO-p15INK4b mice (100%), compared with that obtained in both the MSV-Rgr (10%) and KO-p15INK4b (8.2%) mouse lines, clearly demonstrates the synergistic cooperation between p15INK4b and the Rgr oncogene in vivo.
It has been described that inhibition of p15INK4b expression appears to be a common event in human lymphoid tumors and mouse T-cell lymphomas (37, 38, 39, 40, 41) . Here, the important role of p15INK4b in lymphomagenesis is confirmed. Furthermore, the results indicate that the role of p15INK4b in tumorigenesis is dependent on the concurrent oncogenic activation of various components of the Ras pathway. Indeed, splenic lymphomas are found in 50% of MSV-RGR/KO-p15INK4b mice, whereas this tumor type is found in <1% of KO-p15INK4b mice, which only show extramedullary hematopoiesis. In accordance with these observations, similar results have been obtained when crossing transgenic mice expressing an N-Ras oncogenic variant with p15INK4b-KO mice.4 Finally, the results derived from the study of the CD4-RGR tumors constitute not only additional evidence in favor of the role of alterations in both genes in lymphomagenesis but also a new and significant observation about the collaboration of the two of them in this kind of tumor.
The INK4b gene and the closely related gene, INK4a, are located at the same chromosomal region in both the human and mouse genome, a hot spot for cancer-associated deletions (37 , 42) . Although the inactivation of p16INK4a has been clearly associated with cancer development and progression (43) , the role of p15INK4b as a tumor suppressor gene is still controversial. Therefore, our data constitute new and strong evidence in favor of the critical role of p15INK4b inactivation in the development and progression of some tumor types.
Critical Role of Rgr Oncogenic Activation in T-Cell Tumorigenesis.
An important conclusion derived from this work is the role of Rgr in lymphomagenesis. As it has been described above, MSV-RGR mice lacking p15INK4b showed a remarkable increase in splenic B-cell lymphoma incidence. These results are remarkable, considering that Rgr expression in the spleen of these transgenic mice is modest (data not shown). CD4-RGR transgenic mice provided additional confirmation of the role of Rgr in lymphomagenesis. The expression of Rgr under control of the CD4 promoter induces thymic lymphomagenesis (Fig. 6)
. The fact that NrasT transgenic mice develop thymic lymphomas (44)
is consistent with our previous results in vitro, in which the transforming activity of Rgr is mediated predominantly by Ras.
The study of preneoplastic CD4-RGR thymi produced important insights regarding the Rgr-induced alterations in T cells. In the CD4-RGR transgenic line with higher incidence of tumors and shorter latency, 6-week-old mice showed a significantly altered pattern of their thymocyte subpopulations (Fig. 7)
. The reduction in the number of CD3+high, CD4+CD8–, and CD4–CD8+ cells indicated an increase in the number of undifferentiated thymocytes. Moreover, these cells became more activated, as demonstrated by the levels of expression of specific activation markers (CD25 and CD69). The most interesting results derived from the analysis of the CD44 marker. CD44 is a multistructural and multifunctional cell surface molecule that has been involved in many cellular functions from cell proliferation to cell migration and angiogenesis. In this case, both CD4-RGR transgenic mice lines (lines 42 and 37) showed a similar and substantial increase in the number of CD44+ thymocytes. Interestingly, CD44 has been associated with cancer (45)
, and it has been proposed as a good candidate to predict prognosis in patients with lymphoma (46)
. Our data suggest that CD44 overexpression could be a candidate marker for early detection of Rgr-induced lymphomagenesis.
Recently, the human orthologue of Rgr, hrgr, has been associated with human T-cell malignancies (7) . Similar to rabbit Rgr, which was found as one of the components of a fusion protein isolated from a rabbit squamous cell carcinoma, truncation of hrgr confers transforming properties to its cDNA. DNA rearrangements, which are frequent events in T-cell lymphomagenesis (47) , could have resulted in hrgr truncation. Indeed, a DNA rearrangement has been found within the hrgr gene in a human anaplastic large cell lymphoma cell line (DHL; Ref. 7 ). It is interesting to point out that chromosomal translocations frequently have been associated with sarcomagenesis (48) , and in many cases, these genetic translocations affect a chromosomal region close to the one in which hrgr is located (22q11.2). Therefore, there is a need for additional studies on the role of hrgr in human sarcomagenesis. According to our preliminary results, DNA rearrangements involving Rgr provoke changes in its levels of expression. In normal cells and tissues, the levels of expression of both Rgr and hrgr are low, whereas in transformed cells and tumor samples, a significant level of expression of these genes is detected (3 , 7) . In this work, overexpression of oncogenic Rgr is sufficient to induce the development of fibrosarcomas and lymphomas. Thus, our previous in vitro model is corroborated, in which a mutation affecting the regulation of gene expression, which increases its expression from negligible levels, is the basis for Rgr acting as an oncogene (6) . However, neoplastic alterations have not been detected in some of the tissues in which Rgr has been overexpressed. For example, the MSV-RGR mice express substantial levels of Rgr in liver, brain, and kidney (data not shown), and pathological alterations have not been detected in those tissues after analysis of these transgenic mice at age > 2 years. Therefore, these results suggest that the Rgr-induced transforming phenotype is dependent not only on the expression levels of this gene but also on certain tissue-specific factors. These still uncharacterized elements might be the reason for oncogenic Rgr involvement in lymphomagenesis and, to a lesser extent, in the development of fibrosarcomas.
Taken together, these results reinforce the role of Rgr as an oncogene in different tumor types. In some of the tumors (fibrosarcomas), Rgr-induced tumorigenesis is substantially potentiated by the effect of other genetic alterations, such as a constitutive lack of p15INK4b expression. However, in other cases (lymphomas), expression of Rgr in T cells induced a severe alteration of thymocyte differentiation and strengthens the notion that Rgr plays an important role in lymphomagenesis. Therefore, the high incidence of lymphomas and fibrosarcomas in the transgenic lines and the fact that hrgr also is associated with human lymphomas support this as a model to study sarcomagenesis and lymphomagenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Angel Pellicer, Department of Pathology and New York University Cancer Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016. Phone: 212-263-5342; Fax: 212-263-8211;
4 I. Pérez de Castro, M. Malumbres, M. Benet, M. Jiménez, J. F. García, M. Barbacid, A. Pellicer. Effect of p15INK4b deficiency on N-ras mediated oncogenesis, manuscript in preparation. 
Received 10/28/03. Revised 5/16/04. Accepted 7/ 6/04.
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