Retinoblastoma-Related P107 and Prb2/P130 Proteins in Malignant Lymphomas: Distinct Mechanisms of Cell Growth Control1

Vol. 5, 4065–4072, December 1999 Clinical Cancer Research 4065

Retinoblastoma-related p107 and pRb2/p130 Proteins in Malignant Lymphomas: Distinct Mechanisms of Cell Growth Control1

Lorenzo Leoncini, Cristiana Bellan, ated according to the Kaplan-Meier method and the log- Antonio Cossu, Pier Paolo Claudio, Stefano Lazzi, rank test. We found a positive correlation between the per- Caterina Cinti, Gabriele Cevenini, centages of cells positive for p107 and proliferative features such as mitotic index and percentage of Ki-67(؉) and cyclin -Tiziana Megha, Lorella Laurini, Pietro Luzi, A(؉) cells, whereas such correlation could not be demon Giulio Fraternali Orcioni, Milena Piccioli, strated for the percentages of pRb2/p130 positive cells. Low Stefano Pileri, Costantino Giardino, Piero Tosi, immunohistochemical levels of pRb2/p130 detected in un- and Antonio Giordano2 treated patients with NHLs of various histiotypes inversely Institute of Pathologic Anatomy and Histology, University of Sassari, correlated with a large fraction of cells expressing high Sassari, Italy [L. L., A. C.]; Institute of Pathologic Anatomy and levels of p107 and proliferation-associated proteins. Such a Histology [C. B., S. L., T. M., L. L., P. L., P. T.] and Institute of pattern of protein expression is normally observed in con- Thoracic and Cardiovascular Surgery and Biomedical Technology tinuously cycling cells. Interestingly, such cases showed the [G. C.], University of Siena, Siena, Italy; Departments of Pathology, highest survival percentage (82.5%) after the observation Anatomy, and Cell Biology, Jefferson Medical College, and Sbarro Institute for Cancer Research and Molecular Medicine, Philadelphia, period of 10 years. Thus, down-regulation of the RB-related Pennsylvania 19107 [P. P. C., A. G.]; Department of Scienze pRb2/p130 protein could be one of the reasons why these Odontostomatologiche e Maxillo-Facciali, Universita´degli Studi di cases display such a high rate of proliferation and why they Napoli “Federico II,” Napoli, Italy [P. P. C., C. G.]; Institute of respond so well to therapy. Normal and Pathologic Cytomorphology, Consiglio Nazionale delle Ricerche, Bologna, Italy [C. C.]; and Institute of Hematology, “L.&A. Seragnoli,” Hemolymphopathology Unit, University of Bologna, INTRODUCTION Bologna, Italy [G. F. O., M. P., S. P.] In recent years, the combined use of genetic and biochemical approaches has allowed us to identify regulatory mecha- ABSTRACT nisms controlling cell cycle and cell growth (1, 2). A variety of experiments have documented the critical role of the RB3 pro- pRb/p105, p107, and pRb2/p130 compose the retino- tein family pRb/p105, pRb2/p130, and p107 in controlling the blastoma (RB) family of proteins and regulate cellular G checkpoint of the cell cycle (3). These proteins share con- growth and differentiation. Because recent functional stud- 1 siderable sequence homology and also the ability to interact ies have indicated that the expression of the RB-related with and regulate E2F transcriptional activity (4, 5). proteins p107 and pRb2/p130 are tightly cell cycle regulated, Although pRb/p105, pRb2, and p107 interact with mem- we were interested in investigating their expression along bers of the E2F transcription factor family and have similar with cellular kinetic characteristics and proliferative fea- functional properties, each protein has a different temporal tures of non-Hodgkin’s lymphomas (NHLs). p107 and pRb2/ profile of interaction with different E2F members (6, 7). p130 expression was determined immunohistochemically in Whereas pRb (p105) is found in both quiescent and proliferating biopsy specimens from 83 untreated patients with NHLs of cells, the expressions of pRb2 and p107 are peculiar during the various histiotypes. The expression of these two RB-related cell cycle (8). The binding of pRb2 to E2Fs is detected predom- proteins was correlated with the mitotic index, apoptotic inantly during G , whereas that of p107 is detected during the index, and percentages of Ki-67(؉), cyclin A(؉), p34(؉), 0 G and S phases (9). Both depend on different upstream signals and cyclin B(؉) cells. The overall survival rate was evalu- 1 such as cyclin/CDK complexes or viral oncoproteins (10, 11). The flexibility of this pathway can explain the distinct activities of the three proteins in the regulation of cellular division and cellular differentiation (12). The three proteins exhibit different Received 4/26/99; revised 8/25/99; accepted 8/31/99. The costs of publication of this article were defrayed in part by the growth-suppressive properties in specific cell lines, suggesting payment of page charges. This article must therefore be hereby marked that although the different members of the RB protein family advertisement in accordance with 18 U.S.C. Section 1734 solely to may complement each other, they are not completely function- indicate this fact. ally redundant (13, 14). Although pRb (p105) has been exten- 1 Supported by Sbarro Institute for Cancer Research and Molecular Medicine; by NIH Grants R01 CA 60999-01A1, PO1 NS 36466, and sively shown to play a key role in the negative regulation of PO1CA 56309-06 (to A. G.); a Ministero dell’Universita´ e Ricerca Scientifica e Technologic grant (to L. L.); and an Associazione Italiana per la Ricerca sul Cancro grant (to S. P.). P. P. C. is the recipient of a fellowship from the Associazione Leonardo di Capua, Italy. 2 To whom requests for reprints should be addressed, at Pathology, 3 The abbreviations used are: RB, retinoblastoma; pRb, RB protein Anatomy, and Cell Biology, Kimmel Cancer Institute, Sbarro Institute product; pRb2, pRb2/p130; CDK, cyclin-dependent kinase; NHL, non- for Cancer Research and Molecular Medicine, Thomas Jefferson Uni- Hodgkin’s lymphoma; MI, mitotic index; AI, apoptotic index; B-CLL, versity, 1020 Locust Street, Room 226, Philadelphia PA 19107. Phone: B-cell chronic lymphocytic leukemia; MZL, marginal zone lymphoma; (215) 503-0781; Fax: (215) 923-9626; E-mail: [email protected]. ALCL, anaplastic large cell lymphoma.

Table 1 Clinical characteristics of patients Negative controls were obtained by replacing the primary anti- Number of patients 83 bodies with normal mouse serum. Normal human tonsils served Age in years (median-range) 54–76 as positive controls. Sex (female/male) 46/37 Immunostaining of the cell cycle regulator proteins exam- Location (nodal/extranodal) 73/10 ined in this study was carried out as described above. We used Clinical stage I/II/III/IV 17/21/16/29 anti-p34cdc2 serine/threonine kinase/cdk AB-1 (clone A17.1.1; dilution, 1:100) for the detection of p34, anti-cyclin B-1 AB-1 (clone V152; dilution, 1:100) for the detection of cyclin B, and anti-cyclin A (clone 6E6; dilution, 1:100) for the detection of cellular proliferation (15, 16), little is known about pRb2 and cyclin A. These mouse monoclonal antibodies were obtained p107 in human neoplasias. from Neomarkers (Fremont, CA). The polyclonal antibodies Because of the importance of pRb2 and p107 in controlling anti-Rb2 and anti-p107 have been described previously (10, 12) cellular growth (8, 9), we investigated their expression in rela- and used at the working dilution of 1:1000. Immunostaining for tion to the cellular kinetic characteristics and proliferative fea- Ki-67 with the MIB-1 antibody was performed as described tures of NHLs. We used diffusely growing NHLs of different histopatho- previously (24). logical types as a model system because the NHLs exhibit a Cell Counts and Registration of Mitotic and Apoptotic distribution of mitotic figures and apoptotic cells/bodies that Index. In all sections, cells exhibiting a positive immune approximates regularity better than other neoplasias (17) and are reaction to each antibody were counted in 30 representative classified into disease entities that respect not only the pheno- fields (using a Leitz microscope with a Planapo 100/1 objec- type but also cytogenetic alterations (18). tive), and the results were expressed as percentages of all neoplastic cells in those areas. In selecting the fields for evalu- MATERIALS AND METHODS ation, the following criteria were applied: (a) their representa- tiveness of the tumor’s histiotype; (b) the substantial homoge- Case Selection and Conventional Histopathology. We neity of the neoplastic population examined; and (c) the exact retrospectively studied biopsy specimens from 83 untreated location and content of the reactive cells as revealed by previous patients with various types of NHLs, collected from the Insti- phenotyping in paraffin sections (25). Intra- and interobserver tutes of Pathology of the University of Siena and Bologna. Because comparison of histologically defined lymphomas was reproducibility of counts was 95%. The intensity of immune not the primary goal of this investigation, no attempt was made reactions, i.e., the amounts of reaction product, was estimated to select cases for comparable group size. Cases were chosen on qualitatively. the availability of sufficient amounts of diffuse or diffusely Because mitotic figures and apoptotic cells/bodies can be growing parts of NHL tissue in the paraffin blocks, absence of best recognized in semi-thin sections (26), we re-embedded part extended tissue necrosis, and adequate clinical information, of the lymphoma tissue into Epon-Araldite resin (Monojel, ␮ especially regarding staging and survival. Follow-up data were Kidare, Ireland) and stained 1 m section with Azur A for each available for all patients, with a median follow-up duration of 65 case. MI and AI were registered in each case as described months (range, 24–120). Patients who died from causes other previously (24). Briefly, we used an oil immersion objective than lymphomas were considered as lost to follow-up evalua- (ϫ100) and first established the cellularity in 20 randomly 2 tion, and therefore, their survival periods were censored at the chosen high-power fields, 56,000 ␮m /field, and then assessed date of death. All patients were treated with chemotherapy the MI and AI by registering the percentages of neoplastic cells and/or radiotherapy according to established protocols (19–21). in the mitosis or apoptosis of an entire section/case (100 high- The clinical characteristics of lymphoma cases studied are sum- power fields). With this approach, the relative number of apop- marized in Table 1. Biopsy materials from diseased tissues, totic cells/bodies correlated with the percentages of cells show- obtained at different hours of the day, were immediately sliced ing in situ end-labeling of DNA strand breaks (27). Intra- and and fixed in a neutral, buffered 4% formaldehyde solution (pH interobserver reproducibility of counts was 95%. 7.4) for 24 h and embedded in paraplast at 56°C. For conven- Statistical Analysis. A linear correlation analysis was tional histology, 4-␮m sections were stained with H&E, performed to quantify the strength of the association among all Giemsa, and Gomori’s silver impregnation. Diagnoses were of the parameters examined. For this purpose, the Pearson made independently by four experienced pathologists, based on correlation coefficient r, was computed and evaluated together the REAL classification (18). A final agreement, with the with its statistical significance in terms of error of probability P knowledge of immunohistochemical findings, was reached in (28). Correlation coefficients were also examined, together with every case. scatterplots and regression lines, for a graphical inspection of Immunohistochemical Procedures. Immunophenotyp- the effective linear association between variables at different ing on paraffin sections was performed using a large panel of ranges of values. antibodies to detect formalin-resistant epitopes of malignant To investigate the existence of different populations on the lymphoma cells and the EnVisionTMϩ/HRP method (Dako, basis of variables p107 and pRb2 and to locate cutoff values, the Milan, Italy; Ref. 22). For antigen retrieval, deparaffinized sec- probability density functions were estimated by using the kernel tions of 3–4 ␮m were treated by microwaves or by pressure method (29). This is a nonparametric method that does not cooking in 1 mM EDTA (pH 8.0), for 5 min, followed by cooling impose any presupposed functional forms on the probabilities, at room temperature prior to incubation with the antibodies (23). for example the hypothesis of normality, and estimates proba-

Fig. 1 Immunostaining with anti-cyclin B (a), anti-p34 (b), anti-cyclin A (c), anti-p107 (d), and anti-pRb2 (e). Cyclin B and p34 showed either cytoplasmic or cytoplasmic and nuclear positivity, whereas cyclin A, p107, and pRb2 were found localized in the nucleus. EnVisionTMϩ/HRP method, ϫ350.

bility density functions directly from the sample data. Given a performed with the SPSS statistical package, release 5.0.1 sample of N data points, the density function is estimated (SPSS, Inc., Chicago, IL). through the superimposition of N identical kernel functions centered on each of the data points. We can smooth out the estimate by choosing different forms for the kernel function. For RESULTS example, rectangular functions lead to the classical histogram Qualitative Histological and Immunohistochemical Ob- representation. The major problem with the kernel-based meth- servations. In Azur A-stained semithin sections, mitotic fig- ods is the choice of kernel width. When the width is too large, ures and apoptotic cells/bodies were easily identified (22). Signs the estimated density is oversmoothed, and a possible multimo- of disturbed cell division, such as chromosome bridges, de- dal nature of the distribution is lost. Conversely, a too small tached chromosomes, pyknomitoses, and asymmetrical mitoses, width gives a noisy estimation of the distribution. We chose a could be detected in only a very small fraction of mitotic figures. kernel of triangular form because it allows the width (basis of These were quite evenly distributed, although some focal accu- the triangle) of the kernel function to be estimated as a suitable mulations did occur. More than one-half of the apoptotic cells compromise between noise and smoothing. To do that, a max- exhibited a single, dark-stained, roundish mass with an effaced imum likelihood technique was used. A great advantage of the chromatin structure; the others appeared as apoptotic bodies kernel method is that, when a multimodal probability density with two or more nuclear fragments. Cells positively stained for function is found, cutoff values between different populations p34 or cyclin B showed immune reaction over the cytoplasm are unequivocally identified to correspond at the distribution and in part over the nucleus, whereas the localization of Ki 67, minima. Cutoff values were used to form homogeneous subject cyclin A, and p107 was essentially nuclear (Fig. 1, a–d). Cells groups. stained for pRb2 showed a predominantly nuclear/or perinuclear Survival was evaluated according to the Kaplan-Meier positivity in the majority of the cases (Fig. 1e), but in a few method (30) and the log-rank test (31). Data analysis was cases, a diffuse cytoplasmic stain was also observed.

Fig. 2 Scattergrams derived from plots of MI and AI for each case Fig. 4 Scattergrams derived from plots of percentages of pRb2(ϩ) against the respective fractions of p107- and pRb2-positive cells. The cells for each case against the respective fraction of Ki67(ϩ), cyclin percentages of p107(ϩ) cells were directly correlated with MI and AI, A(ϩ), p34(ϩ), and cyclin B(ϩ) cells. The percentages of pRb2(ϩ) cells whereas a rather loose inverse correlation was found between the showed a weak inverse correlation with the percentages of Ki-67(ϩ), percentages of pRb2(ϩ) cells and both MI and AI. cyclin A(ϩ), and p34(ϩ) cells but not with the percentages of cyclin B(ϩ) cells.

Fig. 3 Scattergrams derived from plots of percentages of p107(ϩ) cells for each case against the respective fraction of Ki67(ϩ), cyclin A(ϩ), Fig. 5 Comparison of percentage of p107(ϩ) cells with the fraction of p34(ϩ), and cyclin B(ϩ) cells. The percentages of p107(ϩ) cells were pRb2(ϩ) cells for each case showed a significant, although moderate, significantly correlated with the percentages of Ki-67(ϩ) and cyclin inverse correlation between the two proteins. Top and right side of the A(ϩ) cells; a similar correlation was also found between p107 staining plot reports the probability density functions (p.d.f.) of the variables for and the percentages of p34(ϩ) and cyclin B(ϩ) cells. which the cutoff values were chosen.

Correlation of p107 and pRb2 with Cellular Kinetic On the other hand, a rather loose inverse correlation was Features. The percentages of p107(ϩ) cells were directly found between the percentages of pRb2(ϩ) cells and both MI correlated with MI (r ϭ 0.72, P Ͻ 0.001) and AI (r ϭ 0.68, P Ͻ (r ϭϪ0.35, P Ͻ 0.001) and AI (r ϭϪ0.35, P Ͻ 0.001) as 0.001) as shown in Fig. 2, a and b. shown in Fig. 2, c and d.

Table 2 Distribution of NHL cases according to the REAL Classification among groups with distinct p107/pRb2 expressiona Histiotypeb p107ϩ/pRb2ϩ p107Ϫ/pRb2ϩ p107Ϫ/pRb2Ϫ p107ϩ/pRb2Ϫ No. of patients surviving B-CLL, MZL 12 (7) 7 MCL 1 5 (1) 1 FCL 1 (1) 21 (9) 5 (3) 2 (2) 15 DLBCL 14 (5) 4 (1) 3 (2) 6 (5) 13 BLL 2 (1) 1 PTCL 2 2 1 (1) 1 ALCL 2 (2) 2 Total 18 (6) 44 (18) 8 (5) 13 (11) a The number of patients surviving for each histiotypes in groups with different p107 and pRb2 expression is reported in parentheses. b MCL, mantle cell lymphoma; FCL, follicular center cell lymphoma; DLBCL, diffuse large B-cell lymphoma; BLL, Burkitt’s-like lymphoma; PTCL, peripheral T-cell lymphoma.

Table 3 Distribution of pathological stage of NHL cases among groups with distinct p107/pRb2 expression p107ϩ/ p107Ϫ/ p107Ϫ/ p107ϩ/ Stage pRb2ϩ pRb2ϩ pRb2Ϫ pRb2Ϫ I2924 II 6 8 3 4 III 5 7 1 3 IV 5 20 2 2 Total 18 44 8 13

Correlation of p107 and pRb2 with Proliferative Fea- tures. The percentages of p107(ϩ) cells were significantly correlated (P Ͻ 0.001) with the percentages of Ki67(ϩ)(r ϭ 0.81) and cyclin A(ϩ) cells (r ϭ 0.72). A similar correlation, although less significant, was also found between p107 staining and the percentages of p34(ϩ)(r ϭ 0.46) and cyclin B(ϩ) cells Fig. 6 Overall survival in relation to p107/pRb2 expression. The (r ϭ 0.44). The percentages of pRb2(ϩ) cells in each case p107ϩ/pRb2Ϫ cases showed the highest survival rate after an observa- showed a weak inverse correlation with the percentages of tion period of 10 years. Ki-67(ϩ)(r ϭϪ0.41, P Ͻ 0.001), cyclin A(ϩ)(r ϭϪ0.32, P Ͻ 0.01), and p34(ϩ)(r ϭϪ0.29, P Ͻ 0.01) but not with the percentage of cyclin B(ϩ) cells. Figs. 3 and 4 show the scatterplots together with regression pression. The different histiotypes were represented in at least lines. It is evident from Figs. 3 and 4 that, although statistical two or more groups, with the exception of B-CLL and MZL, Ϫ ϩ correlation does exist, several cases are remarkably distant from which were present only in p107 /pRb2 cases and BLL and ϩ the regression line. ALCL, which were present only in p107 /pRb2- cases. Patients Correlation between p107 and pRb2. A linear inverse with different stages were included in each group. correlation was found between the percentages of p107(ϩ) and Response to Therapy and Survival Analysis. Kaplan- pRb2(ϩ) cells (r ϭϪ0.45, P Ͻ 0.001), even if the scatterplot Meier cumulative survival curves relative to the four groups are in Fig. 5 clearly shows that some points representing individual reported in Fig. 6. Although the variety of analyzed histological cases are again remarkably distant from the regression line. This types of NHL, the diverging therapy, and the retrospective finding prompted us to further investigate whether cases with nature of the present study precluded an unequivocal assessment distinct expression of p107/pRb2 could be identified. To test this of actuarial survival and disease-free survival, it is of interest ϩ Ϫ hypothesis, the nonparametric kernel method (29) was then that the p107 /pRb2 cases showed the highest percentage of applied, and cutoff values, corresponding to 50 and 25% of cells survival (82.5%) after the observation period of 10 years inde- positive for p107 and pRb2, respectively, were determined on pendently from the histiotype and stage. The comparison of the basis of the estimated probability density functions (Fig. 5). survival curves by the log-rank test (31) gave a P of 0.09. Four groups could then be identified in Fig. 5: values of p107 Ͼ50% and pRb2 Ͼ25% (p107ϩ/pRb2ϩ); values of p107 DISCUSSION Ͻ50% and of pRb2 Ͼ25% (p107Ϫ/pRb2ϩ); values of p107 Recent functional studies of p107 and pRb2 indicated that Ͻ50% and pRb2 Ͻ25% (p107Ϫ/pRb2Ϫ); and values of p107 although the RB family members may be able to complement Ͼ50% and of pRb2 Ͻ25% (p107ϩ/pRb2Ϫ). each other, these proteins are not completely functionally re- Tables 2 and 3 illustrate the distribution of histiotypes and dundant (4). Although p107 specifically interacts with E2F4 and pathological stages among groups with distinct p107/pRb2 ex- E2F5, as does pRb2, the constant presence of the E2F/p107/

cyclin A/CDK2 complex in proliferating cells (9) suggests that antiproliferative and tumor-suppressive potentials in vivo and p107, rather than acting exclusively as a tumor suppressor in vitro using a tetracycline-dependent overexpression sys- protein (32), may also play a positive role in cellular prolifera- tem (43, 44). Alterations in the p107 gene have also been tion (33). These findings confirm the presence of multifunc- identified in a limited number of human hematological ma- tional domains on the nuclear RB products, which may allow lignancies (45). Furthermore, it should be considered that complex interactions with cellular transcription machinery (34). checkpoint control is usually performed by more than one The expression of p107 and pRb2 in malignant lymphomas system. These and other influences make it virtually impos- presented here are in accordance with these experimental data sible to interpret the present results in terms of molecular and support the concept of their different functions in cellular events. growth control. In fact, we found that the percentages of cells Nevertheless, previous reports have shown a tight inverse positive for p107 correlated in a linear fashion with proliferative correlation between tumor malignancy and pRb2 expression in features such as MI and the percentages of Ki-67(ϩ) and cyclin several human neoplasia, suggesting a direct involvement of A(ϩ) cells, whereas such correlation could not be demonstrated pRb2 in the course of those diseases (46–49). In this study, we for the percentages of pRb2(ϩ) cells. present data showing a distinct expression of p107 and pRb2 On the basis of the experimental data, a model for pRb2 among histological types of malignant lymphomas. B-CLL and and p107 control during cell proliferation has been proposed MZL are represented only in the p107Ϫ/pRb2ϩ group of cases, ϩ previously (33). In quiescent G0 cells, the E2F-pRb2 complex whereas BLL and ALCL are represented only in the p107 / is responsible for the active repression of a number of cellular pRb2Ϫ cases. Conversely, the other histiotypes are represented promoters, including those of E2F1, E2F2, and p107 (33–35). in at least two or more groups with different p107 and pRb2 After the release into the cell cycle, pRb2 is phosphorylated expression levels. In particular, this pattern pertains to follicular

by G1 CDKs and subsequently degraded through a protea- cell lymphoma and diffuse large B-cell lymphoma and suggests some-dependent mechanism (36). pRb2 protein level thus the possibility of different pathogenetic mechanisms within drops dramatically to almost undetectable levels, resulting in these types of lymphoma. Our results emphasize the REAL the derepression of a variety of genes, including p107 (32, 34, Classification recommendation to examine each disease entity 37). The accumulated p107 protein is then able to interact with respect to possibly divergent molecular features, growth with E2F4 and E2F5 that have been released from the pRb2 patterns, and clinical aggressiveness (18). Interestingly, the complex and associate with cyclin A/CDK2 (38). Thus, in an p107ϩ/pRb2Ϫ cases showed the highest survival rate (82.5%) ideally controlled proliferating cell population with identical after the observation period. These cases demonstrated low cell cycle and cycle phase times, during which a certain values of pRb2 that inversely correlated with a large fraction of protein can be detected by the correspondent antibody, the cells expressing high levels of p107 and proliferation-associated percentages of cells expressing p107 and pRb2 should be proteins. Such a pattern of protein expression is observed when

inversely related. Our finding of an inverse correlation be- cells enter G1 after mitosis in continuously cycling cells (50). tween the percentages of positive cells for p107 and pRb2 Thus, down-regulation of the RB-related pRb2 protein in ma- supports these observations. However the scatter plot in Fig. lignant lymphomas can be in part responsible for an enhanced 5 clearly shows that some points representing individual cellular proliferation (51) and a better response to therapy (52, cases are remarkably distant from the regression line, indi- 53). It would be extremely interesting to look at the pRb2 cating that we were dealing with considerable variation. expression in a uniform group of patients to determine whether There are many tentative explanations for the deviation of this can be an additional marker for prognosis. individual cases from the ideal line. Many factors may be Unfortunately, few data are known about the transcrip- altered in neoplastic cell populations; the interaction between tional, posttranslational, and regulatory events that involve the individual proteins and the E2F family members and the RB-related proteins and E2Fs, so that little more than specula- timing of formation of particular E2F complexes during the tion can be made at this time. Although complex analysis of cell cycle can be deregulated (39), or a functional interaction important genetic pathways are technically challenging, they are with cyclins may be disturbed because of an overexpression now possible and can provide powerful genetic, cellular, and and/or an unscheduled expression of these latter molecules biochemical approaches to determine the basic mechanism of (40). Yet, proteins with lost or diminished function(s) may cellular growth control. still be detectable by immunostaining (41), and circadian variation in the nuclear expression of cyclins has been re- REFERENCES ported (42). Genetic alterations disrupting the nuclear local- 1. Puri, P. L., MacLachlan, T. K., Levrero, M., and Giordano, A. The ization of RB2 in human cell lines and primary tumors have intrinsic cell cycle: from yeast to mammals. In: G. Stein, R. Baserga, A. 4 been documented recently. Such mutations may reduce Giordano, and D. Denhardt (eds.), Cell Cycle and Growth Control, pp. growth inhibition, because RB2 has been shown to have 15–79, New York: Wiley-Liss, 1999. 2. Hutchison, C., and Glover, D. M. Cell Cycle Control. Oxford: IRL Press at Oxford University Press, 1995. 3. Paggi, M. G., Baldi, A., Bonetto, F., and Giordano, A. The retinoblastoma proteins family in cell cycle and cancer. J. Cell. Biochem., 62: 4 C. Cinti, P. P. Claudio, C. M. Howard, L. M. Neri, Y. Fu, L. Leoncini, 418–430, 1996. P. Tosi, N. M. Maraldi, and A. Giordano. Genetic alterations disrupting 4. Claudio, P. P., Howard, C. M., Baldi, A., De Luca, A., Fu, Y., the nuclear localization of the retinoblastoma-related gene RB2/p130 in Condorelli, G. L., Sun, Y., Colburn, N., Calabretta, B., and Giordano, A. human cell lines and primary tumors, Cancer Research, in press, 2000. p130/pRb2 has growth suppressive properties similar to yet distinctive

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Lorenzo Leoncini, Cristiana Bellan, Antonio Cossu, et al.

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