Cooperative alterations of Rb pathway regulators in mouse primary T cell lymphomas

I. Pérez de Castro1,2,3, M. Malumbres1, J. Santos2, A. Pellicer1 and J. Fernández-Piqueras2

1 Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA and
2 Laboratorio de Genética Molecular Humana, Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alterations in the Rb pathway have been described in many different tumors. In order to study this cell cycle regulatory mechanism in murine T cell lymphomas, we have analyzed the RNA and protein expression of the cyclin D1, cdk4 and retinoblastoma genes in primary tumor samples. We have detected overexpression of the cyclin D1 gene and deficient expression of the retinoblastoma gene in 42 and 28% of these tumors, respectively. The immunohistochemical analysis showed that these RT–PCR results are correlated with a significant increase in the number of positive cells for cyclin D1 and a moderate decrease in the expression of Rb protein, respectively. The analysis of cyclin D1, Rb, p15INK4b and p16INK4a showed that 75% of lymphomas had alterations in these genes and indicates that the Rb pathway is frequently altered in mouse primary T cell lymphomas. Moreover, 31% of lymphomas presented simultaneous alterations in at least two of these genes, suggesting the importance of concurrent alteration of different Rb pathway regulators. In addition, we have characterized these samples for mutational status of the N-ras and K-ras genes. We have only detected mutations in codon 12 of K-ras in six of 49 lymphomas (12%). Interestingly, five of these lymphomas also showed alterations in at least one of the Rb pathway regulators analyzed here. Taken together, these data suggest that deregulation of the Rb pathway regulators and/or oncogenic activation of K-ras may represent a common important clue in progression of murine T cell lymphomas.

Abbreviations: DAB, diaminobenzidine; NMU, N-methyl-N-nitrosourea; TLSR1, thymic lymphoma suppressor region 1.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Murine T cell lymphomas induced by carcinogenic agents, such as {gamma}-irradiation and N-methyl-N-nitrosourea (NMU) treatment, is an important animal model useful in the search for and characterization of genes involved in human lymphoma development. The most frequent genetic alterations described in this model system are activation of the N-ras and K-ras oncogenes (14), trisomy of chromosome 15 and translocation of chromosomes 1 and 5 (4,5). These alterations occur early in disease development and have been reported in {gamma}-radiation- and NMU-induced T cell lymphomas, with the exception of the 1:5 translocation, which is only detected in {gamma}-radiation-induced lymphomas. In contrast, p53 mutations are much less frequent (68).

We have reported specific allelic losses on mouse chromosome 4 in {gamma}-radiation-induced primary thymic lymphomas of C57BL/6JxRF/J F1 (B6RFF1) mice (9). Allelotype analysis of chromosome 4 showed the existence of a critical region of deletion located between markers D4Wsm1 and D4Mit9, named thymic lymphoma suppressor region 1 (TLSR1), as the likely site of one or more putative tumor suppressor genes implicated in murine thymic lymphomas.

TLSR1 is syntenic with the human region 9p21–22 (1011), which is frequently deleted in several types of human tumors (see refs 12–14 as representative examples). The human p16INK4A and p15INK4B genes are candidate tumor suppressor genes for this human region. Homozygous deletion in the 9p21 region is one of the most common types of inactivation of p16INK4a and in 80% of the cases involves both the p16INK4A and p15INK4B genes (15). p16INK4a point mutations and small deletions are also frequent in some tumors (16), but intragenic p15INK4B mutations are very infrequent. Inactivation of these genes often results from promoter region hypermethylation in different types of human tumors (1721).

Recently, we performed a detailed analysis of the p16INK4a and p15INK4b genes in mouse primary thymic lymphomas induced by {gamma}-irradiation (22). De novo methylation of the 5' CpG islands of the p15INK4b and p16INK4a genes was found to be very frequent and correlated with deficient expression of the corresponding mRNA. Our results showed the importance of allelic losses and CpG island methylation for p15INK4b and indicated that this gene is the main target for hypermethylation in {gamma}-radiation-induced T cell lymphomas with independence of p16INK4a status. Similar results have been reported in human hematological malignancies. Different authors have pointed out the frequent and selective methylation of p15INK4B in T cell acute lymphoblastic leukemia (21,23,24). On the other hand, hypermethylation of p16INK4A is more frequent in non-Hodgkin's lymphoma (23). Thus, all these results indicate the involvement of the p15INK4B and p16INK4A genes in lymphoid tumors and support an important tumor suppressor role for these genes in hematological malignancies.

The products of the p15INK4B and p16INK4A genes have been shown to arrest cell cycle progression in the G1 phase (25,26). These INK4 proteins inhibit phosphorylation of the retinoblastoma protein (pRb) by D-type cyclins (D1, D2 and D3) and cyclin-dependent kinases 4 and 6 (Cdk4 and Cdk6) (27). Phosphorylation of pRb by the cyclin–CDK complexes leads to release of the transcription factor E2F in an active form and contributes to the G1 to S transition. Lack of the INK4 proteins and pRb and/or overexpression of active cyclin–CDK complexes may induce a constitutive progression from G1 to S, which has been reported to be an important contributing factor to cancer development (28,29).

Although it is clear that growth factor signaling is required to pass through the G1/S transition, the intracellular signaling pathways that lead to activation of the cell cycle machinery have not been well established. However, the ras proto-oncogene products, main components of signal transduction pathways, act to couple external signals to the cell cycle machinery (30). Thus, constitutive activation of ras genes may contribute to cell cycle alteration.

In this work we have extended previous analyses of the p16INK4a and p15INK4b genes (22) to others regulators of the cell cycle (the Cdk4, cyclin D1, Rb, K-ras and N-ras genes) to further evaluate the full extent of involvement of the Rb pathway and its upstream regulators in {gamma}-radiation-induced T cell lymphomas. The results suggest that the cooperative effects of deregulation of these Rb pathway regulators may represent a common alteration in the progression of murine T cell lymphomas.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice and induction of tumors
Interstrain crosses, involving males from strain RF/J and females from strain C57BL/6J, were made to generate F1 hybrid mice, named B6RFF1. C57BL/6J and RF/J mice were purchased from the Mouse Resource of The Jackson Laboratory (Bar Harbor, ME). Thymic lymphomas were induced in 4-week-old mice using weekly whole body {gamma}-radiation (four times, 175 rad/exposure) (9). All 49 tumor samples analyzed in this study were primary T cell lymphomas in the most advanced stage of T cell lymphoma development (Stage III).

DNA and RNA isolation
Mice were killed and the tissue was stored at –80°C. Genomic DNA isolation was performed using standard procedures. Total RNA was isolated from tissues using the FastRNA kit (BIO 101, Vista, CA). DNA contamination was removed by digestion with DNase I.

Mutational analysis of N-ras, K-ras and cdk4
We performed a mismatched PCR–RFLP analysis to detect point mutations in codons 12, 13 and 61 of the N-ras and K-ras genes (31,32). This procedure allowed us to detect all possible mutations in the two first bases of each codon. H-ras has not been analyzed, since this gene is only expressed at low levels in thymus (33) and there is no evidence in favor of its oncogenic activation in lymphomas (3).

Primers and conditions for the analysis of each codon were modified from those previously described by Mangues et al. (34; Table IGo). Amplifications were performed in a volume of 25 µl with a final concentration of 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 1–1.5 mM MgCl2, 200 µM each deoxynucleotide triphosphate (dATP, dCTP, dGTP and dTTP), 0.2 µM each primer, 0.1% Triton X-100 and 1 U of polymerase (DynaZymeTM; Finnzymes Inc., Espoo, Finland). Two hundred nanograms of genomic DNA as template were used in each PCR reaction. The Mg2+ concentrations used for each codon are given in Table IGo. PCR amplifications were carried out in a model 2400 DNA thermal cycler (Perkin Elmer-Cetus, Norwalk, CT) with cycling times of 94°C for 10 min (one cycle) and 35 cycles of 1 min at 94°C, 1 min at 55°C and 30 s at 72°C, followed by a final incubation at 72°C for 10 min and then cooling at 4°C.


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Table I. Primers, restriction endonucleases and expected fragment sizes for mismatched PCR–RFLP analysis of K-ras and N-ras
 
The endonuclease digestions of PCR products were performed using the conditions recommended by the manufacturer (New England Biolabs, Beverly, MA). DNA fragments obtained after the digestion were separated by electrophoresis on 3% agarose gels and detected by ethidium bromide staining. All uncut samples were sequenced to confirm and characterize the potential mutation (Sequenase DNA sequencing kit; US Biochemical, Cleveland, OH).

A PCR–RFLP analysis was used to detect point mutations in the two first bases of codon 24 of the cdk4 gene. The genomic sequence of cdk4 was amplified by PCR with the primer pair CD4-R and CD4-F (Table IIGo). The PCR products were digested with AvaI using the conditions recommended by the manufacturer (Boehringer Mannheim, Indianapolis, IN). The sequence of the wild-type amplified fragment contains only one restriction site for AvaI in codon 24. Thus, the wild-type sequence is cut by the endonuclease, while the mutated sequence remains uncut. The digested fragments were electrophoresed in 1.5% agarose gels and were visualized by ethidium bromide staining.


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Table II. Primer sequences, conditions and expected fragment sizes for RT–PCR analysis of the Cyl-1, cdk4 and Rb genesa
 
Murine Cyl-1, Rb, cdk4 and ß-actin probes: isolation and labeling
Mouse Cyl-1, Rb, cdk4 and ß-actin probes were obtained from non-treated C57BL/6J mouse by RT–PCR. The cDNA from each mRNA was generated using the primers and conditions described below (see also Table IIGo). These PCR products were isolated from agarose gels and subcloned in pCR II using the TA Cloning kit (Invitrogen, San Diego, CA). The identity and integrity of the insert was confirmed by sequencing.

Inserts were labeled with digoxigenin using a random primed labeling kit (Boehringer Mannheim).

Expression studies by RT–PCR
Five hundred nanograms of total cellular RNA were used to generate cDNA in a reaction containing 200 µM dNTPs, 50 mM Tris–HCl, pH 8.3, 6.0 mM MgCl2, 40 mM KCl, 4.0 mM dithiothreitol and a hexanucleotide mixture (Boehringer Mannheim). This mixture was heated to 90°C for 1 min and 64°C for 1 min. One unit of AMV reverse transcriptase (Finnzymes) was added and the samples were incubated at 42°C for 1 h. An aliquot of 1 µl of this reaction was used for co-amplification of the ß-actin and either Cyl-1, Rb or cdk4 cDNAs in a reaction mixture containing 0.4 µM each primer (Table IIGo), 200 µM dNTPs, 1.5 mM Mg2+ and 1 U of DNA polymerase (DynaZymeTM, Finnzymes). Amplification was carried out in a Perkim Elmer-Cetus DNA thermal cycler 2400, in a final volume of 25 µl, according to the conditions described in Table IIGo. Primers of Rb and cdk4 genes were designed from previously reported cDNA sequences (35,36). ß-actin primers were supplied by Clontech (Mouse ß-Actin Control Amplimer Set; Clontech Laboratories, Palo Alto, CA) and Cyl-1 primers were previously described by Bianchi et al. (37). Reactions were followed by a final incubation at 72°C for 5 min. Under the above conditions, all the amplification reactions were in the linear range. Primers for ß-actin were always used as an internal control in the RT–PCR.

RT–PCR products were run in agarose gels and detected by ethidium bromide staining (Rb assays) or by blotting and subsequent hybridization with specific probes using the Digoxigenin Labeling and Detection System (Boehringer Mannheim) (Cyl-1 and cdk4 assays). PCR products were quantified by densitometric analysis (1D Analysis and Hand Scanner Settings; Biomed Instruments, Zeineh Programs). cDNAs from RNA of 16 normal thymus B6RFF1 mice were amplified in order to determine the expression pattern from unaffected tissue. To assess the expression levels of each gene we used the ratio between the internal control (ß-actin) signal and the Rb, cdk4 or Cyl-1 signals, respectively. The mean and standard deviation (SD) of normal tissue expression were calculated and compared with tumor expression of each sample. Decreased expression was considered when the values of the tumoral samples were >2 SD and overexpression when the values were <2 SD.

Immunohistochemistry
Immunohistochemistry was performed on formalin fixed, paraffin embedded tissues using an automated immunohistochemical stainer (NexES; Ventana Medical Systems; Tucson, AZ). Briefly, tissues sections 5 µm thick were baked at 60°C overnight and deparaffinized to water (two xylene washes, followed by two washes through graded 100%, 95% and 50% alcohols). Antibodies against pRb and cyclin D1 were obtained from Pharmingen (San Diego, CA) (mouse monoclonal, 1:10 dilution) and Zymed (San Francisco, CA) (rabbit polyclonal, clone AM29, 1:10 dilution), respectively. Heat-induced antigen retrieval was performed in citrate buffer prior to application of the primary antibodies. Incubation was overnight with secondary detection with diaminobenzidine (DAB). Samples were considered negative for the expression of both proteins if <10% of the cells were labeled.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cyl-1, Rb and cdk4 expression
We have previously reported the importance of p15INK4b and p16INK4a inactivation in {gamma}-radiation-induced T cell lymphomas (22). These two genes arrest cell cycle progression in the G1 phase. In the present study we have extended this previous analysis to other Rb pathway components. A wide panel of murine induced T cell lymphomas have been analyzed for altered expression of Cdk4, cyclin D1 and pRb by RT–PCR.

Expression analysis of the cdk4 gene showed the same band intensity in control thymus and T cell lymphomas, indicating no alteration in cdk4 expression in these tumors (Figure 1aGo). However, as shown in Figures 1b and 1cGo, expression levels detected for Rb and Cyl-1 were altered in some tumors. Whereas Rb and Cyl-1 cDNA products presented normal levels in the control tissues, 13 of 45 (28%) lymphomas exhibited a decrease in mRNA expression of Rb and 20 of 47 (42%) lymphomas showed significant overexpression of D1 cyclin (Table IIIGo). Although we did not have available material from these tumor samples for an immunohistochemical analysis, we have performed this assay in some equivalent lymphomas of C57BL/6JxBalb/c F1 mice. These {gamma}-radiation-induced primary lymphomas show similar phenotypic and genetic alterations to those analyzed in this work by RT–PCR (38). Immunohistochemical analysis of normal thymus showed that this tissue is negative for cyclin D1 expression (Figure 2Go). In contrast, a significant increase in the number of positive cells was detected in the selected cases which showed cyclin D1 overexpression by RT–PCR. On the other hand, the Rb antibody stained the majority of cells of normal thymus, whereas the percentage of positive cells was lower in the samples in which decreased expression was detected by RT–PCR (Figure 2Go). However, all these lymphomas were considered positive because >10% of their nuclei were labeled.



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Fig. 1. Expression analysis of Cyl-1, cdk4 and Rb genes. The first two lines represent control DNA samples from B6RFF1 hybrid (F1) mice. The remaining lines represent different tumour samples in each panel. (a) Expression analysis of cdk4 did not show any differences between control thymus and T cell lymphomas. (b) Lines 3, 4, 6 and 7 show overexpression of cyclin D1 (Cyl-1). (c) In lines 3 and 7 expression of Rb was decreased.

 

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Table III. Rb pathway alterations and K-ras mutational status in {gamma}-radiation-induced thymic lymphomas in B6RFF1 micea
 


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Fig. 2. Immunohistochemical analysis of Cyl-1 and Rb. Immunhistochemical staining of a normal thymus (1 and 2) and four representative samples of lymphomas of C57BL/6JxBalb/c F1 mice (3–6). The normal thymus shows only occasional cells stained with the cyclin D1 antibody (1) and a high percentage of Rb-positive cells (2). Samples 3 and 6 show overexpression of cyclin D1 (Cyl-1) and a decreased number of Rb-positive cells, respectively. Samples 4 and 5 show normal expression levels of both proteins.

 
Considering expression of the p15INK4b and p16INK4a genes in the same tumors (22) (Table IIIGo), of the 44 lymphomas that could be simultaneously evaluated for decreased expression of p15INK4b, p16INK4a and Rb and for overexpression of cyclin D1, 33 (75%) showed some of these alterations. In these 33 lymphomas the pattern of alterations was: only one gene affected in 19 (43% of the total); two genes altered in 11 (25% of the total); alterations in three genes in two lymphomas (4% of the total); all these alterations in one tumor (2% of the total) (Table IIIGo). Of the 19 lymphomas that had one of these alterations, overexpression of cyclin D1 was detected in eight (18% of the total), decreased expression of Rb in six (13% of the total), decreased expression of p15INK4b in four (9% of the total) and decreased expression of p16INK4a in only one (2% of the total). Among the tumors with at least two of these alterations, the most frequent situation was overexpression of Cyl-1 and decreased expression of p15INK4b (five lymphomas, 11% of the total).

Mutational analysis of the K-ras, N-ras and cdk4 genes
DNA from the 49 primary T cell lymphomas was screened to detect mutations in codons 12, 13 and 61 of the K-ras and N-ras genes. Mismatched PCR–RFLP analysis only showed mutations in codon 12 of K-ras in six of 49 lymphomas (12%) (Figure 3Go and Table IIIGo). All these samples, with the exception of R14A, showed only the mutant allele, indicating loss of the wild-type allele. Five of these lymphomas carried G:C->A:T transitions in the second base of codon 12 of K-ras, changing Gly to Asp, which is the most frequent mutation detected in ras genes in thymic lymphomas (2,3,34). In tumor R3A, we found a G:C->A:T transition in the first base of this codon that changed Gly to Ser. This mutation has not been previously detected in this type of tumor.



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Fig. 3. Analysis of K-ras, N-ras and cdk4 mutations in representative samples of lymphomas. (a) Mismatched PCR–RFLP analysis of K-ras and N-ras. Non-mutated samples show a wild-type digested band while mutated samples (lines 3, 6 and 7 for codon 12 of K-ras) show an undigested band of higher molecular weight. Line 1 for codon 61 of N-ras corresponds to an undigested band of a control F1 sample. All mutated lymphomas lost the wild-type allele with the exception of line 7. (b) Partial sequence amplified with primers CD4-F and CD4-R in the PCR–RFLP analysis of codon 24 of cdk4. The wild-type sequence contains an AvaI site that includes codon 24 (underlined) and that was used to detect point mutations in the first two bases of this codon. (c) Genotyping for the cdk4 mutations. All samples show a wild-type digested band, with the exception of line 1 that corresponds to an undigested band of a control B6RFF1 hybrid.

 
We screened for the presence of a cdk4 mutation in codon 24 that converts this molecule into a dominant oncogene (39). This alteration implies an Arg->Cys exchange and generates a dominant oncogene that is resistant to normal physiological inhibition by p16INK4A. In our work none of the 49 T cell lymphomas analyzed showed mutations in this codon (Figure 3Go).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study we have compared mRNA expression of five members of the Rb pathway (p15INK4b, p16INK4a, cyclin D1, cdk4 and Rb), the most frequently disrupted in cancer cells (28), in {gamma}-radiation-induced mouse T cell lymphomas. We have also characterized the mutational status of the N-ras and K-ras genes, whose oncogenic activation have been implicated in several human tumors (40), in the same tumor samples.

Amplification-induced overexpression of Cdk4 has been reported in glial cell lines (41), human sarcoma cell lines (42), NMU-induced primary rat mammary tumors (43) and human and mouse intestinal carcinogenesis (44). However, our present findings indicate that cdk4 expression is not altered in {gamma}-radiation-induced primary T cell lymphomas. Moreover, the existence of an activating point mutation in codon 24, like that reported in a human melanoma by Wölfel et al. (39), can be ruled out in murine T cell lymphomas since none of the analyzed lymphomas showed mutations in this codon.

In contrast, the Cyl-1 gene (mouse cyclin D1 gene) was overexpressed in 42% (20/47) of the primary T cell lymphomas analyzed in this study. It is interesting to note that Cyl-1 shows very weak expression in normal T cells, as demonstrated in the control samples used in this work and in other studies (45). Thus, the presence of a greater number of non-T cells in T cell lymphomas could be the explanation for this abnormal expression of cyclin D1 in T cell lymphomas. We have ruled out this possibility since we have detected complete loss of heterozygosity in these primary lymphomas using microsatellite polymorphisms amplified by PCR (9). Thus, contamination of non-tumoral cells seems not to be a contributing factor. Moreover, T cell lymphomas, like those analyzed in this study, consist mainly of a monoclonal expansion from a single T cell (4). Taken together, these data favor the idea that cyclin D1 overexpression detected in the T cell lymphomas analyzed here might be attributed to the tumor T cells. However, the strongest evidence was obtained in the immunohistochemical analysis of {gamma}-radiation-induced lymphomas of C57BL/6JxBalb/c F1 mice that are equivalent to those analyzed in this work. Thus, as Figure 2Go illustrates, cyclin D1 overexpression was contributed by T cells rather than macrophages, blood vessels, fibroblasts or other cell types within the tumor.

Mouse cyclin D1, located on mouse chromosome 7 (36), has been related to murine neoplasias. Overexpression of cyclin D1 was detected in early mouse skin carcinogenesis (37,46), intestinal adenomas from multiple intestinal neoplasia (Min) (44) and lymphomas induced by Friend murine leukemia virus (45). In the T cell lymphomas analyzed here we ruled out the induction of cyclin D1 overexpression by trisomy of chromosome 7 or translocations that involved the region where Cyl-1 is located (data not shown). So, the principal mechanism that induces overexpression of cyclin D1 in this type of tumor is still an open question.

In this study we have detected decreased expression of Rb in 42% of lymphomas as derived from the RT–PCR ratios obtained in co-amplification of Rb and ß-actin cDNAs. The immunohistochemical analysis shows that the low expression of Rb detected by RT–PCR correlated with a decrease in the number of Rb-positive cells (Figure 2Go). However, this analysis did not show any lymphoma completely negative for Rb and it remains unclear whether the decrease in expression of Rb detected in this work by RT-PCR and immunohistochemical analysis constitutes a significant loss of function of this protein with a key role in the origin and development of mouse T cell lymphomas.

One of the most interesting conclusions of our results is derived from simultaneous analysis of the Rb pathway regulators characterized here. The functional connections among Rb, p15INK4b, p16INK4a and cyclin D1 imply that abrogation of the Rb pathway through alteration of any of these four proteins might produce similar effects upon tumor progression. Thus, inactivation of either the Rb, p15INK4b or p16INK4a genes or overexpression of cyclin D1 might eliminate the G1/S checkpoint. In the present study, we have detected alterations in some of these genes in 75% of tumors. Interestingly, although the Rb alterations were excluded, this percentage could be greater because some alterations (e.g. point mutations that alter the phosphorylation status of the protein or post-translational aberrations) escape the RT–PCR analysis that we have used in this work. Thus, our results indicate that the Rb pathway is frequently altered in mouse primary T cell lymphomas.

Of the different patterns of alterations that we have detected for these four Rb pathway components, the most frequent was alteration of only one of these genes (43% of lymphomas). These results are in agreement with the hypothesis that implies a Rb pathway operating as a functional unit (47). On the other hand, our results also provide evidence for a possible collaborative role of alterations of these genes. At least two of these alterations were detected in 31% of lymphomas. The most frequent situation was coincidence of overexpression of cyclin D1 and deficient expression of p15INK4b (five of 14, 11% of the total). Different laboratories have reported similar cooperative models between the components of the Rb pathway for its participation in human tumorigenesis. Alterations of the p16INK4A gene with aberrations of the cyclin D1 gene were reported for the first time by Lukas et al. (48) in different cancer cell lines and then in cell lines from squamous cell carcinomas of the head and neck (49) and in mantle cell lymphomas (50). Concurrent overexpression of cyclin D1 and Cdk4 has been detected in murine and human intestinal neoplasias (44). Nielsen et al. (51) have found inactivation of the retinoblastoma protein associated with deregulation of cyclins D1 and E in primary breast tumors. Finally, Jadayel et al. (52) have suggested that overexpression of cyclin D1 and inactivation of p15INK4B and p16INK4A may characterize certain cases of mantle cell non-Hodgkin's lymphomas, whereas Grønboek et al. (53) extended these concurrent alterations to p53 and Rb in the same type of lymphomas.

In summary, it is clear in view of this evidence that in some tumor types alteration of only one of the Rb pathway components is not enough to deregulate the cell cycle in favor of tumoral progression. In contrast to human studies, no detailed analysis of these genes has been carried out in murine primary tumors. Belinsky et al. (54) showed that Rb, cyclin D1 and p16INK4a did not cooperate in the progression of mouse lung tumor, but they only analyzed the protein levels of these three genes in a small number of samples (six tumors). On the other hand, it is worth noting that, to the best of our knowledge, no previous study in mouse models has reported concurrent alterations of different members of the Rb pathway. Thus, this study represents the first report on the simultaneous involvement of some of the components of the Rb pathway in murine carcinogenesis.

The discoveries of the last few years in the field of cyclin-dependent kinases and their regulators and targets have provided us with a better understanding of the complexity of the control mechanisms more directly implicated in progression of the cell cycle. However, the majority of the signaling components between external signals and the cell cycle machinery are still not well known. Ras proteins could be upstream regulators of the cyclin-dependent kinases. In support of this hypothesis, Winston et al. (30) have suggested that ras-activated signal transduction pathways may link growth regulatory signals from the cell surface to the cell cycle machinery via modulation of G1 cyclin expression. Other laboratories have proposed that Rb (55), cyclin D1 and p27KIP1 (56) are critical targets of the ras signaling cascade. In our study we have detected mutations only in K-ras codon 12, which is in agreement with previous analysis of ras genes in mouse lymphomas induced by chemical carcinogens or radiation (2,3,8,34,57). Interestingly, five of the six lymphomas with mutations in K-ras also showed alterations in at least one of the Rb pathway regulators. The main alteration detected together with a K-ras mutation was cyclin D1 overexpression (four of six lymphomas). This is of particular interest since it reinforces the hypothesis that cyclin D1 can be one of the mediators of the transforming action of activated ras (58). These findings could also provide evidence in support of cooperation between these two genes, as previously suggested by Lovec et al. (59) in different tumor types, including B cell lymphomas, or in a way similar to other cooperating proteins, such as, for example, pRb and p53 (60), p53 and p16INK4A (61) or p16INK4A and ras (62). It is worth noting that one lymphoma (R9b) that had K-ras mutated did not show alterations in any of the four Rb pathway components analyzed and it may indicate that oncogenic activation of K-ras could also affect a Rb independent pathway.

In summary, the main conclusion that can be drawn from our studies is that alterations in the components of the pRb pathway and/or oncogenic activation of K-ras appear to play a key role in the origin and progression of murine T cell lymphomas. These findings widen our understanding of the multistep progression implicated in mouse T cell lymphomagenesis. However, nine of 49 lymphomas did not show any of the genetic alterations analyzed here. Thus, further work is need to clarify the complex mechanism involved in the development of murine thymic lymphomas.


    Acknowledgments
 
We thank Mark Pendfold for review of the manuscript and Dr Herman Yee for technical support in the immunohistochemical analysis. These studies were funded by a grant from the Fundacion Ramón Areces (Madrid, Spain) and grants PM 96/001 (Ministerio de Educacion y Cultura, Spain) to J.F.-P. and CA36327 from the NIH to A.P.


    Notes
 
3 To whom correspondence should be addressed at: Department of Pathology and Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA Email: perezi01{at}popmail.med.nyu.edu.. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 18, 1998; revised April 9, 1999; accepted May 27, 1999.