Oncogene (1999) 18, 3501 ± 3510 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Prohibitin, a potential tumor suppressor, interacts with RB and regulates E2F function

Sheng Wang1, Niharika Nath1, Matthew Adlam2 and Srikumar Chellappan*,1

1Department of Pathology, College of Physicians and Surgeons, Columbia University, 630W 168th Street, New York, NY 10032, USA; 2Department of Microbiology and Immunology, College of Physicians and Surgeons, Columbia University, 630W 168th Street, New York, NY 10032, USA

The retinoblastoma tumor suppressor protein and its tion or apoptosis (Chellappan, 1994; Kohn, 1998; Lam family members, p107 and p130, are major regulators of et al., 1998; Lam and La Thangue, 1994; Wiman, the mammalian cell cycle. They exert their growth 1993). suppressive e€ects at least in part by binding the E2F The retinoblastoma tumor suppressor protein, Rb, family of transcription factors and inhibiting their and its family members p107 and p130 have been transcriptional activity. Agents that disrupt the interac- shown to be the major modulators of the G1/S tion between Rb family proteins and E2F promote cell transition in mammalian cells (Dyson, 1994; Ewen, proliferation. Here we describe the characterization of a 1994). In normal mammalian cells, their function is novel interaction between Rb family proteins and a regulated by phosphorylation mediated by cyclin- potential tumor suppressor protein, prohibitin. Prohibitin dependent kinases at precise stages of the cell cycle physically interacts with all three Rb family proteins in (Sherr, 1994). It has been clearly established that vitro and in vivo, and was very e€ective in repressing cyclins D and the asssociated kinases cdk4 and cdk6 E2F-mediated transcription. Prohibitin could inhibit the are involved in the phosphorylation of Rb and its activity of E2Fs 1, 2, 3, 4 and 5, but could not a€ect the consequent inactivation during G1 phase of the cell activity of promoters lacking an E2F site. Surprisingly, cycle, permitting the transition to S phase (Weinberg, prohibitin-mediated repression of E2F could not be 1995). Cyclin E in conjunction with cdk2 also has been reversed by adenovirus E1A protein. A prohibitin mutant reported to inactivate Rb (Duronio et al., 1998; that could not bind to Rb was impaired in its ability to Lundberg and Weinberg, 1998; Taya, 1997), but cyclin repress E2F activity and inhibit cell proliferation. We E/cdk2 can induce cell proliferation independent of the believe that prohibitin is a novel regulator of E2F Rb protein also. In the case of p107 and p130 the activity that responds to speci®c signaling cascades. picture is not very clear, but apparently cyclins D, E and their associated kinases are involved in their Keywords: Rb; E2F; cell cycle; transformation; tumor regulation (Beijersbergen et al., 1995; Xiao et al., suppression 1996). In addition, Cyclin A/cdk2 has been implicated in the inactivation of the p107 protein (Hauser et al., 1997; Faha et al., 1992; Shirodkar et al., 1992). This scenario is supported by the ®ndings that Rb physically Introduction interacts with D-type cyclins whereas p107 binds to cyclins E and A (Ewen et al., 1992, 1993; Kato et al., The progression of the mammalian cell cycle is 1993; Lees et al., 1992; Meyerson et al., 1992). precisely regulated by a combination of stimulatory Though phosphorylation is the main mechanism of and repressive elements (Bishop, 1991; Marshall, 1991). inactivating Rb and its family members in normal The complex cell-cycle machinery mainly consists of mammalian cells, they can be activated by additional cyclins, cyclin dependent kinases and their inhibitors, means. For example, inactivation of the Rb gene by down-stream targets of the cyclin-dependent kinases mutation or deletion is widespread in a variety of which inlcude the Rb family of proteins and their human cancers (Cox et al., 1994; Cropp, 1995; Lee et down-stream targets, mainly the E2F family of al., 1988). In addition to mutation, many viral transcription factors (Adams and Kaelin, 1996a; oncoproteins like the E1A of adenovirus, large T- Cobrinik, 1996; Grana and Reddy, 1995; Kamb, antigen of SV40 and the E7 protein of human 1995; Sherr, 1994; Sherr and Roberts, 1995; Slansky papilloma virus have been found to physically interact and Farnham, 1996; Weinberg, 1995). It has been with Rb family proteins leading to their functional demonstrated that inappropriate activation of any of inactivation equivalent to phosphorylation (Nevins, the positive acting elements or inactivation of the 1992, 1993, 1994; Whyte et al., 1989). In addition to negative acting components can lead to neoplasia cyclins and viral oncoproteins, interactions with other (Hamel and Hanley-Hyde, 1997; MacLachlan et al., cellular proteins contribute considerably to the 1995; Pines, 1995; Sutherland et al., 1995). The function of the Rb family tumor suppressors (Taya, components of the cell cycle machinery can respond 1997). The E2F family of transcription factors are to the appropriate extra-cellular signals and respond in especialy notable in this context, since they are believed the proper fashion facilitating proliferation, differentia- to be the major down-stream targets of Rb proteins (La Thangue, 1996a; Nevins, 1992). Earlier observations had suggested that Rb protein *Correspondence: S Chellappan Received 1 September 1998; revised 20 November 1998; accepted physically interacts with E2F and represses its 13 January 1999 transcriptional activity (Bagchi et al., 1991; Chellap- Prohibitin-mediated regulation of E2F function SWanget al 3502 pan et al., 1991; Hiebert et al., 1992), and this is a binding domain was used as a bait to screen a HL60 major component of Rb-mediated growth regulation cDNA/VP16-AD library as previously described (Qin et al., 1995; Beijersbergen and Bernards, 1996). It (Junaief et al., 1994; Vojtek et al., 1993). Out of 80 is now apparent that Rb binds to the transcriptional clones that were obtained in the screen, 26 were activation region of E2F (Helin et al., 1992; Kaelin et partially sequenced. Among the known genes that were al., 1992), and the Rb-mediated inhibition of E2F obtained, the most interesting appeared to be a involves histone de-acetylases (Brehm et al., 1998; Luo sequence corresponding to amino-acids 1 ± 157 of et al., 1998; Magnaghi-Jaulin et al., 1998). The Rb human prohibitin. We chose to focus our attention family members show a certain degree of preference to on this gene, since it has been reported to have tumor di€erent E2Fs, with Rb binding to E2Fs 1, 2 and 3 and suppressive and anti-proliferative e€ects. p107 and p130 binding to E2Fs 4 and 5 (Beijersbergen The same two-hybrid system was used to evaluate and Bernards, 1996; Cobrinik, 1996). Many of the whether the interaction was speci®c (Figure 1). A full genes that are required for cell cycle progression length prohibitin cDNA was cloned into the pGAD- require E2F, and the over-expression of E2F1 has NOT vector as a VP16 AD fusion protein (Vojtek et been found to induce S phase entry (Adams and al., 1993) and was screened for interaction with Kaelin, 1996b; Johnson, 1993). Repression of E2F by pBTM116-p130. A strong interaction was observed in Rb is believed to prevent the expression of such genes the form of dense colonies, but there were no colonies required for S phase progression, which leads to the observed on plates transformed with the control vector cell cycle block. Indeed, a variety of experiments (pBTM116). All the observed colonies gave a positive strongly suggest that E2F is the main down-stream color reaction for b-galactosidase staining. Interest- target of Rb protein and Rb exerts its growth ingly, prohibitin showed an extremely weak binding to suppressive e€ects to a great extent by inhibiting E2F a mutant p130 carrying a C?F substitution in the activity (La Thangue, 1996; Qin et al., 1995; Weinberg, pocket domain (Claudio et al., 1994, 1996), suggesting 1995). that an intact pocket domain is required for the Earlier studies have shown that E2F function is interaction. As shown in Figure 1d, e and f, prohibitin regulated by Rb family proteins, cyclin A (Adams and interacts with Rb and p107 also quite eciently. Kaelin, 1996a), the dimerization partners of E2F The ability of prohibitin to bind to the Rb family (Bandara et al., 1994; La Thangue, 1994) as well as proteins was examined by an in vitro GST anity the co-activator p300 (Trouche et al., 1996; Trouche chromatography. 35S-labeled prohibitin was synthesized and Kouzarides, 1996). In many cases, the proteins by in vitro transcription-translation in rabbit reticulo- associated with E2F have been found to change during cyte lysates and its ability to bind to GST fusions of cell cycle progression as well as di€erentiation Rb, p107 or p130 was assessed. It was found that (Chellappan, 1994; Corbeil et al., 1995; Ikeda et al., prohibitin could bind eciently to all the three pocket 1996; Strom et al., 1998). Studies from our laboratory proteins (Figure 2a). This experiment along with the had found that two di€erent p130-E2F complexes yeast two-hybrid results suggests that the interaction appear upon the di€erentiation of HO-1 melanoma between prohibitin and the Rb family proteins is cells (Jiang et al., 1995). Similarly, p130-E2F complexes probably direct. Further, prohibitin could not bind to were the predominant E2F containing moieties in GST beads carrying mutations in the pocket domain of di€erentiated U937 cells (Zhang and Chellappan, Rb or p130. In a similar experiment, in vitro 1996). The experiments described in this paper stem synthesized adenovirus E1A, like prohibitin could from our e€orts to identify and clone any novel protein that may interact with p130 or other members of Rb family and regulate E2F function. We ®nd prohibitin, a potential tumor suppressor, to be able to bind Rb, p107 and p130 very eciently and modulate E2F function. Prohibition is a 30kD protein whose gene was originally cloned based on its ability to induce growth arrest in mammalian cells (Nuell et al., 1991). the actual mechanism of prohibitin function is not yet known, and the results described in this paper provide potential mechanisms of prohibitin function. Since prohibitin has been shown to be associated with certain cell-surface receptors like the IgM receptor (Terashima et al., 1994) it could be involved in mediating cell cycle regulation in response to speci®c signal transduction pathways.

Results

Prohibitin interacts with Rb, p107 and p130 in vitro and Figure 1 A two-hybrid analysis of the prohibitin-p130 interac- tion. A full-length prohibitin gene fused to the VP16 transcrip- in vivo tional activation domain was checked for its ability to interact with a control vector (pBTM116; (a)) or the same vector carrying A yeast two-hybrid system was used to screen for a wild-type or mutant p130 gene (b and c). The ability of proteins that interact with human p130 protein. The prohibitin to interact with p130, Rb and p107 was assessed full length human p130 fused to the LexA DNA similarly (d, e and f) Prohibitin-mediated regulation of E2F function SWanget al 3503 bind to Rb, p107 and p130, but not the mutant was no prohibitin present in a control immuneprecipi- proteins. E2F1 on the other hand bound only to GST- tate, where a c-myc antibody was used. These results Rb beads as expected. Under similar conditions of were con®rmed by performing the IP-Western experi- binding, in vitro synthesized JNK1 protein could not ment in the opposite fashion: immunoprecipitation was bind to any of the beads, suggesting that the done using an anti-c-myc antibody as control, or a interactions we observe are speci®c events. These prohibitin antibody, and the presence of Rb, p107 and results support the earlier ®nding that prohibitin p130 examined by Western blotting. As shown in requires an intact pocket domain for interaction, Figure 2c, all the Rb family members could be detected similar to E2F or viral oncoproteins. Prohibitin was in a prohibitin immunoprecipitate, but not in the c-myc unable to dissociate E2F from Rb and Rb containing control. One of the blots was stripped and probed for complexes, when in vitro synthesized prohibitin was the presence of JNK1; we do not ®nd any JNK1 in the added to whole-cell extracts (data not shown). prohibitin IP lane, suggesting that the observed Attempts were made to verify whether prohibitin interactions were speci®c. Co-immunoprecipitation of interacts with the Rb family proteins in vivo in prohibitin with Rb family proteins was also observed mammalian cells. To verify this, whole cell extracts in other cell lines like U937, T47D etc. (data not prepared from Daudi and Ramos cells were immuno- shown). These ®ndings strongly suggest that prohibitin precipiated with antibodies to Rb, p107 and p130. The can interact with proteins of the Rb family in presence of prohibitin in the immune precipitates was mammalian cells, and this interaction can be detected examined by a Western blot analysis. As can be seen without over-expressing any components. Since prohi- from Figure 2b, prohibitin was found to co- bitin was found to bind to all the three pocket proteins, immunoprecipitate with Rb, p107 and p130. There further studies were conducted mainly on Rb.

Figure 2 Prohibitin interacts with Rb family proteins in vitro and in vivo.(a)Anin vitro GST-binding assay showing the binding of 35S-labeled prohibitin to GST fusions of Rb, p107 and p130 (upper panel). RL indicates one ®fth of the amount of reticulocyte lysate used for binding. Prohibitin could not bind to a Rb protein with an exon 21 deletion or a p130 protein with a C?F Figure 3 Prohibitin can repress the transcriptional activity of substitution. Similar assays using 35S-labeled E1A, E2F1 and E2F. (a) T47D cells were transfected with an E2-CAT reporter JNK1 are shown in the lower panels. (b) Prohibitin associates along with an E2F1 expression vector (lanes 2 and 3). Co- with Rb family proteins in vivo. One hundred mg of whole-cell transfection of prohibitin totally represses E2F-mediated tran- extracts from Daudi or Ramos cells were immunoprecipitated scription (lane 3). (b) E1A cannot reverse prohibitin-mediated with antibodies to c-myc (control) Rb, p107 or p130. The repression of E2F. The transcriptional activity of E2F1 in T47D presence of prohibitin in the immunoprecipitates was detected cells was repressed by co-transfecting Rb (lanes 3 and 4) or by a Western-blot analysis. (c) The IP-Western analysis was prohibitin (lanes 5 and 6). Adenovirus E1A could reverse Rb- performed using antibodies to c-myc and prohibitin; the presence mediated repression of E2F activity (lane 4) but had no e€ect on of RB, p107, p130 or JNK1 in the precipitate was examined by prohibitin alone (lane 6) or when prohibitin is co-transfected with Western blotting Rb (lane 7) Prohibitin-mediated regulation of E2F function SWanget al 3504 Prohibitin can repress E2F transcriptional activity which was the internal control for transfection (b-gal values ranged from 0.12 ± 0.14 in T47D cells). This A variety of cellular and viral proteins that bind to the appears to suggest that prohibitin is probably acting on pocket domain of Rb family of proteins can a€ect the E2F speci®cally, and may not a€ect other transcription transcriptional activity of E2F (Chellappan et al., factors or a€ect promoters lacking an E2F binding site. 1992). To examine whether prohibitin is also capable The speci®city of prohibitin-mediated repression was of modulating the function of E2F, we performed a veri®ed by performing additional transient transfection series of transient transfection experiments. As shown experiments. Transfection of an E2-CAT vector in Figure 3a, transient transfection of E2F1 along with carrying mutated E2F sites resulted in no detectable an E2CAT vector induced transcription very eciently CAT activity; in contrast, transfection of an equal in T47D cells (Figure 3, lane 2). Co-transfection of amount of wild-type E2-CAT vector gave rise to high prohibitin totally repressed the transcription induced amounts of CAT activity, showing that the promoter is by E2F1 (Figure 3, lane 3); prohibitin was as ecient being driven by the endogenous E2F transcription as Rb in this aspect. Prohibitin could repress E2F1- mediated transcription in other human cell lines as well (Daudi, Raji, Ramos, ZR75-30, C33A etc; data not shown). Surprisingly, adenovirus E1A could not reverse prohibitin-mediated repression of E2F1 activ- ity (Figure 3b, lane 6). This is in marked contrast to repression of E2F activity by Rb family proteins (Hiebert et al., 1992); as shown in Figure 3b, lane 4, E1A was able to reverse Rb-mediated repression of E2F completely under identical conditions. This might suggest that prohibitin may be repressing transcription mediated by E2F by a mechanism di€erent from the one used by Rb. Supporting this hypothesis, we have not observed a direct binding of prohibitin to the activation domain of E2F1, unlike Rb (data not shown). Interestingly, co-transfection of prohibitin could block the E1A from reversing Rb-mediated expression of E2F activity. As shown in Figure 3b, E1A could block Rb-mediated reversal of E2F (Figure 3b, lane 4), but had no e€ect on prohibitin induced repression (Figure 3b, lane 6). When prohibitin was co-transfected along with Rb and E1A there was no detectable transcriptional activation (Figure 3b, lane 7). This could imply that E1A is unable to reverse Rb-mediated transcriptional inhibition by Rb and prohibitin can withstand the e€ects of E1A. This also raises the possibility that prohibitin may block the E1A-mediated dissociation of E2F from Rb.

Prohibitin can target all transcriptionally active E2Fs Since prohibitin was found to interact with all three Rb-family proteins, we decided to examine its ability to repress transcription mediated by di€erent members of the E2F family. E2Fs 1 ± 5 are known to be transcriptionally active (Slansky and Farnham, 1996) unlike the recently-describe E2F6, which has a mainly repressive function (Trimarchi et al., 1998). We focused our studies on E2Fs 1 ± 5. Co-transfection experiments performed in T47D cells show that prohibitin can repress transcription mediated by all the ®ve di€erent Figure 4 (a) Prohibitin can repress all active E2F family E2Fs (Figure 4a). In contrast, Rb can repress only members. E2-CAT reporter was co-transfected with E2F1, 2, 3, E2Fs 1, 2 and 3 (Figure 4b), as has been described 4 or 5 in the indicated lanes. Co-transfection of prohibitin could previously (Beijersbergen et al., 1994; Cobrinik et al., e€ectively repress all the ®ve E2Fs (lanes 7 ± 11). DP1 was co- 1993). This supports the possibility that prohibitin- transfected along with E2Fs 4 and 5 to obtain optimal transcription. (b) In contrast, Rb could repress onkly E2Fs 1, 2 mediated repression of E2F may involve mechanisms and 3 as reported previously (lanes 6, 7 and 8). (c) Prohibitin that are distinct from the ones used by Rb protein cannot repress promoters lacking an E2F binding site. Co- itself. It is also possible that prohibitin collaborates transfection of prohibitin with an excess of E2-CAT reporter with endogenous p107 and p130 to repress E2Fs 4 and results in a loss of transcriptional activity (lane 4); in contrast, 5, which are not repressed by the Rb protein. prohibitin has no e€ect on co-transfected AP1CAT or c-fos CAT. Transcription from the E2CAT is dependent on the E2F site since Prohibitin did not have any e€ect on transcription a reporter carrying a point mutation in this region has no activity from a pSVbgal vector in any of the experiments, (lane 1). Prohibitin had no e€ect on a pSVb-Gal vector Prohibitin-mediated regulation of E2F function SWanget al 3505 factors (Figure 4c, lane 3). Co-transfection of a of E2F-mediated transcription. The strategy used was prohibitin expression vector totally abolished this to map the region of prohibitin required for binding to E2F site-dependent transcriptional activity, showing Rb and to check whether mutants that cannot bind to that prohibitin could repress the endogenous E2Fs as Rb can inherit E2F function. Amino-terminal trunca- eciently as over-expressed extraneous E2Fs (Figure tions of prohibitin were generated by PCR and the 4c, lane 4). In contrast to E2-CAT, over-expression of resulting genes were transcribed and translated in vitro. prohibitin had no e€ect on the transcriptional activity The ability of the di€erent deletion mutants to bind to of a c-Fos CAT or an AP1-CAT vector (Figure 4c, Rb was checked by a GST-binding assay as described. lanes 6 and 8). Since these two promoters had no E2F As shown in Figure 6a, deletion of the ®rst 33 binding sites, it is likely that the repressive e€ects of amino-acids of prohibitin has no e€ect on its ability to prohibitin are restricted to the E2F transcription bind Rb; similarly, a truncation of up to 74 amino- factors and promoters driven by them. It is also acids did not abolish its Rb-binding. Extending the apparent that the repression of E2F by prohibitin is deletion to 116 residues totally impaired its ability to not mediated by a repression of the basal transcrip- bind to Rb, suggesting that the Rb-binding region tional machinery, since the promoters lacking an E2F spans residues 74 ± 116. This was veri®ed by generating site could function eciently in the presence of a mutant which had an internal deletion of the residues prohibitin. 74 ± 116. As shown in Figure 6a, deletion of these residues eliminated the ability of prohibitin to bind to Rb, suggesting that these residues are indeed necessary Endogenous prohibitin levels a€ect E2F activity The ®nding that co-transfection of prohibitin can a€ect the transcriptional activity of E2F prompted us to examine whetehr the levels of endogenous prohibitin had an e€ect on E2F function. A Western-blot analysis of the levels of prohibitin in ten human breast cancer cell lines identi®ed three (BL15, BL19 and BL20; ZR 57 ± 1, BT474 and DU 4475 respectively) which had high levels of prohibitin (Figure 5a), and three in which prohibitin was absent or extremely low (BL 11, 12 and 16; MDAMB157, MDAMB134VI and BT549 respec- tively). Four cell lines had intermediate levels of prohibitin (BL13, BL14, BL17 and BL18; MDAMB435S, ZR75-30, BT483 and T47D respec- tively). Attempts were made to compare the E2F activities in two cell lines, one having low and the other having high levels of prohibitin. BL15 (ZR75-1) and BL16 (BT549) cells were chosen for this purpose, since they could be transfected eciently. Increasing amounts of E2F1 was co-transfected with 1 mg of the E2CAT reporter in BL15 and BL16 cell lines. It was found that low levels (1, 2 or 3 mg) of E2F1 could not induce the transcription of the E2CAT vector in the BL-15 (ZR 75-1) cells which had high amounts of prohibitin; but 4 mg of E2F1 could eciently induce transcription (Figure 5b). This showed that all the necessary co-factors for mediating transcription by E2F1 are present in the cell. In contrast, co- transfection of even 1 mg of E2F1 could eciently induce transcription in BL-16 (BT-549) cells, which had no detectable prohibitin. We next examined whether prohibitin levels had any e€ect on a promoter that lacked E2F binding sites. Transfection of di€erent levels of AP1-CAT showed that the level of transcription from this promoter was quite comparable in both the cell lines (Figure 5c). This observation supports the earlier ®nding that prohibitin did not repress the AP1-CAT in a co-transfection Figure 5 Endogenous prohibitin levels a€ect E2F activity. (a) assay. It appears that high levels of prohibitin in cells Western blot analysis of prohibitin levels in ten human breast can indeed a€ect transcriptional activity of E2F1 and cancer cell lines; note the high level of prohibitin in BL15 (ZR 75- 1) and very low levels in BL16 (BT-549). (b) Co-transfection of probably the expression of cellular promoters having increasing amounts of E2F1 along with an E2-CAT vector in E2F binding sites. BL15 and BL16 cells. Only the highest amount of E2F1 tested (4 mg) could induce transcription in BL15 cells whereas 1 mgof E2F1 could eciently induce transcription in BL16 cells. (c) Binding to Rb correlates with repression of E2F activity Prohibitin levels do not a€ect the transcriptional activity of an AP1CAT vector. Indicated amounts of AP1CAT were transfected Experiments were designed to assess whether prohibitin into the same cell lines; there was comparable amount of activity binding to Rb correlates with the observed repression at all levels of vector tested Prohibitin-mediated regulation of E2F function SWanget al 3506 for binding to Rb. This span of amino-acids had no prohibitin (Figure 6c). An immunoprecipitation of the LXCXE motif which is present in many Rb-binding extracts with an anti-myc antibody followed by a proteins. An analysis of the sequence showed 45% Western blot analysis using an anti-Rb revealed that identity and 70% similarity to a region of RBP1, a the transfected wild-type prohibitin does indeed known Rb-binding protein. Interestingly, three of the interact with Rb in vivo. There was no Rb present in four point mutations of prohibitin reported in sporadic a myc immunoprecipitate where d74 ± 116 was co- breast cancers fall within this span (Sato et al., 1992, transfected (Figure 6d). Since the two extracts had 1993). comparable levels of Rb and myc-tagged prohibitin, it The ability of the mutant d74 ± 116 to repress E2F- appears that there is a direct correlation between the mediated transcription was examined by transient ability of prohibitin to bind to Rb and to repress transfection assays. As shown in Figure 6b, the transcription. It is likely that prohibitin is also binding mutant that could not bind to Rb could not repress to p107 and p130, as observed in untransfected cells. E2F-mediated transcription either; similar results were obtained with the mutant 116 ± 275 (data not shown). Prohibitin can suppress cell proliferation The correlation between binding to Rb and repressing E2F was veri®ed more directly. Wild-type or d74 ± 116 Prohibitin has been demonstrated to be a strong prohibitin that carried a myc-tag at the amino-terminal suppressor of cell proliferation, but the mechanisms end were generated, and were co-transfected along with involved are not yet clear. Since we observed that Rb into T47D cells. A Western blot analysis of the prohibitin can associated with Rb and express E2F whole-cell extracts from the transfected cells show activity, we decided to examine whether there is a comparable expression of wild-type and mutated correlation between its association with Rb and growth restrictive e€ects. To evaluate this, two di€erent human cell lines, T47D as well as ZR75-30, were stably transfected with a control pSV-NEO vector, or a pCDNAIII-Prohibitin vector, which carries a neomycin resistance marker. After selection in G418 for 14 days, the number of antibiotic resistant colonies was determined. As shown in Table 1, transfection of wild-type prohibitin into T47D cells reduced the number of colonies 3 ± 4-fold, compared to the control vector. Similar results were obtained with ZR75-30 cells (data not shown); the magnitude of repression by prohibitin was comparable to that of Rb under similar conditions. Interestingly, the d74 ± 116 construct was signi®cantly impaired in repressing cell proliferation. Similar results were obtained with the 116 ± 275 mutant also (data not shown). Since these prohibitin mutants have been found to be impaired in Rb binding, it appears that there is a direct correlation between the ability of prohibitin to bind Rb, repress E2F and impart growth arrest.

Table 1 Growth inhibtion by prohibition requires Rb-binding Number of colonies First Second Vectors transfected experiment experiment pSVNeo 196 191 pBASE-Puro 198 199

Rb (0.5 mg) 125 131 Figure 6 Correlation between Rb-binding and prohibitin Rb (1 mg) 69 71 function. (a) A GST binding assay to map the Rb-binding Rb (2 mg) 53 56 region of prohibitin. The indicated mutants were generated in vitro and the binding to Rb tested as in Figure 2a. RL indicates Prohibition, WT (0.5 mg) 131 135 one ®fth of the protein used in the binding assay. (b) A prohibitin Prohibition, WT (1 mg) 71 75 mutant that is unable to bind Rb cannot express E2F. Co- Prohibition, WT (2 mg) 56 53 transfection experiments were performed in T47D cells as in Figure 3a and the ability of a wild-type prohibitin or a prohibitin Prohibition, d74 ± 116 (0.5 mg) 179 175 d74 ± 116 mutant to repress E2F1 activity was assessed. (c) Over- Prohibition, d74 ± 116 (1 mg) 185 188 expression of myc-tagged prohibitin WT or d74 ± 116 mutant in Prohibition, d74 ± 116 (2 mg) 195 196 T47D cells, as examined by a Western blot analysis using an anti- Suppression of colony formation by prohibition requires Rb binding. prohibitin antibody. (d) Prohibitin d74 ± 116 mutant does not T47D breast carcinoma cells grown in 35 mm dishes were stably interact with Rb in vivo. T47D cells over-expressing WT or transfected with the indicated vectors, and selected in antibiotic for mutant prohibitin shown in c were transfected with Rb and the 14 days. Colonies having 20 or more cells were counted. pCMV-Rb whole-cell extracts were immuno-precipitated with an anti-c-myc was co-transfected with pBABE-Puro to impart resistance; pCDNA3- antibody. The presence of Rb in the immune-precipitates was prohibition, WT and d74 ± 116, carried neomycin resistance markers. assessed by Western blotting. The control lane had extracts from Selection was done in Neomycin or Puromycin depending on the untransfected T47D cells vectors transfected Prohibitin-mediated regulation of E2F function SWanget al 3507 Discussion cancers did not identify any mutations, suggesting that prohibitin inactivation may not be a causative step for The studies described in this paper identify prohibitin familial breast cancer but could be playing a role in the as an Rb-binding protein which can modulate E2F generation of sporadic cancers (Sato et al., 1993). Our activity quite eciently. The ®nding as such is results indicate that three of the four prohibition intriguing, since prohibitin itself is strongly anti- mutations reported in exon 4 localize to the Rb proliferative, and is interacting with known growth binding domain we have mapped making it a suppressor molecules. Our ®ndings suggest that possibility that prohibitin cooperates with Rb family interaction with Rb family proteins is probably members to act as a tumor suppressor. necessary for prohibitin to arrest cell proliferation. It is interesting to note that prohibitin can interact Prohibitin was originally cloned based on its ability with all the three Rb family proteins quite eciently to induce growth arrest in mammalian ®broblasts and despite lacking the canonical LXCXE motif which is HeLa cells (McClung et al., 1989; Nuell et al., 1991); present in many Rb binding proteins (Taya, 1997). later studies have shown that prohibitin-mediated Despite the absence of this sequence, prohibitin binds growth arrest is due to a G1/S block (Liu et al., to the pocket domain of Rb, p107 and p130, suggesting 1994; Nuell et al., 1991; Roskams et al., 1993). that probably an LXCXE motif is not essential for Information on the molecular nature of prohibitin is binding to the pocket. This is true for certain other Rb- rather scanty, and it has no discernable functional binding proteins that have been described. In pre- domains (McClung et al., 1995). Prohibitin is highly liminary experiments in U937 cells, we do not ®nd the conserved in a variety of organisms ranging from yeast prohibitin-Rb interaction to be cell cycle regulated. to man. In addition to regulation of mammalian cell Unlike many other pocket-binding proteins, prohibitin cycle, it has been reported that prohibitin plays a role does not have a negative e€ect on Rb function; in in inducing senescence, and can limit the proliferative other words, it does not inactivate Rb like E1A or life span of yeast (McClung et al., 1995). Speci®cally, cyclin D/cdk4. Prohibitin on the other hand can be deletion of the yeast prohibitin gene in haploid cells imagined to enhance Rb function similar to another resulted in an increase of the yeast life span by about Rb binding protein, Brg1 (Junaief et al., 1994). We 30%, whereas its over-expression reduced the life-span have been unable to quantify any synergistic growth by 20% (Franklin and Jazwinski, 1993). suppressive e€ect since both Rb and prohibitin have The potential role of prohibitin in human breast very strong anti-proliferative e€ects by themselves. cancer has been evaluated. Sato et al. (1992) analysed The ®nding that prohibitin can a€ect all the di€erent DNA from 23 sporadic breast tumor samples for E2F family proteins raises many possibilities. First, it is mutations in exons 4 and 5 of prohibitin. The results probably targeting a domain on E2F that is shared by revealed the presence of four point mutations in exon all the di€erent family members. Prohibitin seems to 4; three were in the coding region, and the fourth was di€er in this aspect since Rb shows a preference to in the intron-exon junction (Sato et al., 1992). E2F2 1, 2 and 3 and p107/p130 to E2Fs 4 and 5. Apparently, mutations in the prohibitin gene in exon Second, if prohibitin is repressing E2F activity through 6 have also been detected by a SSCP analysis, though Rb, p107 and p130, it may be able to cover the entire the nature of this alteration is not known (Sato et al., spectrum of E2Fs. This could be one probable 1992). An analysis of 76 familial early-onset breast scenario, since prohibitin did not bind to E2F directly

Figure 7 A model for prohibitin-mediated repression of E2F activity. In a normal situation, adenovirus E1A can dissociate Rb from E2F reversing the repressive e€ects of Rb. Prohibitin binds to a Rb molecule that is bound to E2F and probably recruits a co- repressor. In the presence of prohibitin, E1A is unable to dissociate Rb from E2F or the co-repressor from prohibitin and hence is unable to reverse prohibitin-mediated repression of E2F Prohibitin-mediated regulation of E2F function SWanget al 3508 in an in vitro GST-binding assay. It should be pointed prohibitin was cloned into pPCR-II vector and linearized out, though, that even if prohibitin is inhibiting E2F with XbaI for in vitro transcription using SP6RNA activity through the Rb protein, other mechanisms and polymerase. Prohibitin mutants 33 ± 275, 74 ± 275 and 116 ± domains of E2F may be involved in the repression. 275 were generated by PCR and transcribed similarly. The The precise mechanism by which prohibitin represses internal deletion mutant d74 ± 116 was generated by an overlap-extension protocol and cloned into the same vector. E2F activity is not yet clear. One major di€erence in The resulting mRNAs were translated in rabbit reticulocyte the repression by prohibitin compared to the repression lysates (Promega) in the presence of 35S-methionine (New by Rb family members is that prohibitin-mediated England Nuclear). Eight ml of synthesized polypeptide was repression cannot be reversed by adenovirus E1A incubated with glutathione beads carrying equal amount protein. As has been well-established, Rb binds to the (90 mg) of GST fusion proteins in 200 ml protein binding activation domain of E2F and E1A disrupts this bu€er (20 mM Tris pH 7.5, 50 mM KCl, 0.5 mM EDTA, interaction releasing free, active E2F. In the presence 1mM DTT, 0.5% NP-40, 3 mg/ml BSA) at 48C for 2 h. The of prohibitin such an activation is compromised. It is beads were washed six times with 1 ml protein binding bu€er possible that prohibitin is preventing the E1A-mediated and eluted with 10 mM glutathione. Eluates were separated in dissociation of E2F from Rb; such a scenario is a 10% SDS-polyacrylamide gel and visualized by autoradio- graphy. The protein amount in control input lanes were depicted in the model in Figure 7. Alternately, E1A approximately one-®fth of the total used in binding assay. may also bind to the pocket domain of Rb but allows prohibitin and a potential co-repressor to inhibit E2F. We are currently evaluating the potential role of Yeast Two-hybrid interaction assay additional proteins like histone de-acetylases in the A LexA-p130 fusion cloned into pBTM116 vector was used prohibitin-mediated repression of E2F activity. to screen an HL60 cDNA library in a two-hybrid assay in the There is a good correlation between the ability of yeast strain L40 (His7, Lys7, Leu7, Trp7, Ura7), as prohibitin to bind to Rb, repress E2F activity and previously described (Vojtek et al., 1993; Junaief et al., arrest cell proliferation. This is based on the 1994). To assess the interaction between prohibitin and Rb observation that the prohibitin molecule that lacks family proteins, human p107 cDNA containing the pocket the Rb-binding domain is unable to repress E2F as domain and the C-terminal domain were cloned into well as induce growth arrest. This is quite conceivable pBTM116 vector as a LexA DNA binding domain fusion; pBTM116-Rb was similar, and was a gift from Dr Stephen since E2F1 has been shown to be a strong inducer of Go€. Full length human WT p130 or a point mutant with a cell proliferation and inhibiting its activity could be an C894?F substitution was cloned into pBTM116 as a e€ective means of arresting cell proliferation. It should BamHI ± PstI fragment. Full length human prohibitin gene be pointed out that in the case of Rb all the mutations was cloned into pVP16 as a BamHI ± EcoRI fragment. in the pocket domain which impairs its ability to PBTM116 vectors carry selection markers for Ura and Trp, repress E2F eliminates the ability of Rb to arrest cell and pVP16 for Leu2. Positives for interaction were re- proliferation. Certain point mutations outside the con®rmed by transferring the colonies to a ®lter and pocket domain, though, can abolish the E2F binding performing as an in situ b-galactosidase assay. without compromising the anti-proliferative function. The results described here demonstrate that there are Immunoprecipitation and Western blot analysis molecules that can negatively regulate E2F function in addition to the Rb family proteins. Such molecules Mouse monoclonal antibodies to Rb and c-myc were obtained from Oncogene Science; anti-p107 and -p130 appear to be distinct from Rb in the mechanism of antibodies were from Santa Cruz Biotechnology. The anti- repression of E2F as well as the spectrum of E2Fs they prohibitin rabbit-polyclonal antibody was a kind gift of Dr J can regulate. It is very likely that the involvement of Keith McClung. A goat anti-human IgG antibody was di€erent classes of molecules in the regulation of E2F obtained from Southern Biotechnology Associates and used will enable this transcription factor to respond to a at 1 mg/ml to stimulate Ramos cells. variety of di€erent signaling cascades in an appropriate Whole-cell extracts were prepared by hypotonic shock fashion. For example, it has been reported that followed by salt extraction, as described previously. 50 ± prohibitin is associated with the IgM receptor in rat 200 mg of whole-cell extracts were treated with 5 ml of the B-cells (Terashima et al., 1994); it may be envisaged appropriate primary antibody in a volume of 100 mlat48C that prohibitin may be involved in conducting signals for 1 h. Three mg of Protein A-sepharose or Protein G Sepharose in 100 ml volume was then added and incubated from this receptor. Preliminary results suggest that for additional 1 h. The binding was performed in a bu€er prohibitin can respond to IgM signaling and its e€ects containing 20 mM HEPES pH 7.9, 40 mM KCl, 1 mM MgCl2, on E2F are compromised upon activation of this 0.1 mM EGTA, 0.1 mM EDTA, 0.1 mM DTT, 0.1 mM NaF,

receptor (data not shown). Further studies on 0.1 mM Na3VO4, 0.5% NP40 and 3 mg/ml BSA. The beads prohibitin and the mechanisms involved in its were washed six times with 600 ml of the same bu€er, boiled regulation of E2F will shed light on hitherto unknown in 20 ml of SDS sample bu€er, and separated on 8 or 10% mechanisms regulating cell cycle components. polyacrylamide gels. After semi-dry transfer to supported nitro-cellulose membrane, the blots were probed using the appropriate antibody. The proteins were detected using an ECL system from Amersham. Materials and methods Transient and stable transfections In vitro binding assays T47D human breast cancer cells were transfected by calcium GST fusions of Rb and p107 containing pocket and C- phosphate precipitation method using standard protocols. terminal domains, or the pocket domain of p130 were used in Generally, 2 mg of plasmids were used unless noted otherwise, this study. The mutant GST-Rb lacked exon 21; the p130 and a pSVbgal vector was included in all transfections. mutant had a C894?F substitution. Full length human PCDNA3-prohibitin was generated by sub-cloning a full Prohibitin-mediated regulation of E2F function SWanget al 3509 length cDNA as a BamHI ± XhoI fragment obtained from colonies having 20 or more cells were counted. In all the pCR-II vector; amino-terminal myc-tagged prohibitin was transfections, Rb was co-transfected with a pBABE-Puro generated in a pMum vector and sub-cloned as a EcoRI/NotI vector; Prohibitin constructs carried a Neomycin resistance fragment in pcDNA3 vector. Constructs pDCE2F1, pE2CAT marker. and pSVRb have been described before. Transient transfec- tions were performed in 100 mm dishes for 72 h; assays for CAT and b-galactosidase performed using standard proto- cols. Acknowledgements Stable transfections were performed on 35 mm dishes, We thank Dr J Keith McClung for providing the prohibitin each having approximately 10 000 cells. Selection in 1 mg/ml cDNA and antibodies, as well as for helpful suggestions. of Puromycin or 40 mg/ml neomycin was commenced 48 h This study was supported by a grant from the NCI after transfection. After 14 days of selection, cells were ®xed (CA63136). SPC is a recipient of the Irma-Hirschl Trust in 3.7% formaldehyde, stained with 0.5% crystal violet and Research Award.

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