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[Cancer Research 65, 7223-7230, August 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Mouse Mammary Tumor Virus Env–Derived Peptide Associates with Nucleolar Targets in Lymphoma, Mammary Carcinoma, and Human Breast Cancer

Allan Bar-Sinai1, Nir Bassa1, Maria Fischette2, Michael M. Gottesman3, Dona C. Love4, John A. Hanover4 and Jacob Hochman1

1 Department of Cell and Animal Biology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel; 2 National Heart, Lung, and Blood Institute; 3 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute; and 4 Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland

Requests for reprints: Jacob Hochman, Department of Cell and Animal Biology, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. E-mail: hochman{at}vms.huji.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mouse mammary tumor virus (MMTV) is a type B retrovirus associated primarily with mammary carcinomas in laboratory mice. The best-documented association of MMTV with non–mammary tumors is with T cell lymphomas (1, 2). Previously, we have shown that the leader peptide of the Env-precursor of MMTV is translocated from the cytoplasm to the nucleoli of murine T cell lymphomas (3, 4). This protein, designated p14 based on Western blotting analysis, corresponds to the first 97 NH2-terminal amino acids of the MMTV 73 kDa Env-precursor, terminating just before the beginning of gp52 (ref. 4; see also Fig. 1A). The mass of this protein determined by mass spectrometry is 11 kDa. p14 has been identified and characterized using a monoclonal antibody (M-66) generated against a cell surface epitope of T-25-Adh cells (3). T-25-Adh are nontumorigenic, immunogenic (substrate-adherent) cell variants derived from highly tumorigenic (suspension-borne) T-25 cells (5, 6). T-25 cells were derived from the S49 T cell lymphoma (5). Monoclonal antibody M-66 also identified an additional protein (named p21), which is expressed only in T-25 cells and their derivative sublines (4). Other murine lymphoma cells that harbor MMTV and express p14 are devoid of p21 (3). As p21 is recognized by both M-66 and by an antibody against MMTV-gp52 (3), this protein consists of p14 with an additional 7 kDa fragment of the NH2-terminal region of gp52. This is schematically indicated in Fig. 1A.



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  		Figure 1. p14 is present in mouse mammary carcinomas that harbor MMTV. A, p14 and p21 within the MMTV env precursor protein. B, exponentially growing cells were lysed in SDS-PAGE sample buffer and run on 15% gels, blotted and probed with {alpha}-p14 antibodies as described in Materials and Methods. T-64 and Rev-2-T-6 cells (see Materials and Methods) served as controls expressing p14 and p21, and p14, respectively.

 
Based on colocalization and coimmunoprecipitation studies (4), p14 binds to the shuttling nucleolar protein B23, implicated in both transport and growth regulation, as well as other cellular functions (7, 8). As in the case of B23, actinomycin D induces redistribution of p14/21 from the nucleolus to the nucleoplasm (4). Association with B23 has been previously reported for other auxiliary nucleolar retroviral proteins, such as Rev (HIV) and Rex (human T cell lymphotrophic virus). Indeed, abundant evidence suggests that viruses may target the nucleolus and its components to favor viral transcription, translation, and perhaps alter the cell cycle in order to promote virus replication. For a recent review on the role of the nucleolus/nucleus in viral infections and cancer, see ref. (9).

There has been additional interest in MMTV in recent years because of data associating an MMTV-like retrovirus with human breast cancer (10–13). In these studies, MMTV-like Env gene sequences that were 95% to 100% homologous to mouse MMTV sequences were found in 38% of the analyzed human breast cancers. These sequences have not been detected in normal breast tissue (10, 11, 13). Furthermore, these sequences have also been detected in T cell lymphomas of breast cancer patients who were simultaneously diagnosed with both diseases (10). Liu et al. (12) have recently found a complete proviral MMTV sequence in the genome of human breast cancer tissues. An 86% identity exists between the translated 5'-terminal env sequence (Genbank accession number AF248270) reported by this group with the p14 amino acid sequence (see below).

In the present study, we further characterize p14, as well as identify target proteins that bind to it. We also show that expression of p14 is of more general significance in that it is seen in the nucleoli of murine carcinomas, as well as T cell lymphomas that harbor MMTV. Furthermore, we provide immunohistochemical evidence that a subset of human breast cancer tissues express this protein in their nucleoli.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
S49-derived cell lines were grown in DMEM, with 10% horse serum (Biological Industries, Beit-Haemek, Israel) at 37°C in a humidified atmosphere containing 5% CO2. T-64 cells are highly tumorigenic, suspension-borne derivatives of T-25 cells (3) which express both p14 and p21. Rev-2-T-6 were derived from T-25-Adh cells (14) and express only p14. Mm5MT (ATCC #CRL-1637) and 4T1 (ATCC #CRL-2539) murine mammary carcinoma cell lines (American Type Culture Collection, Rockville, MD) were grown in the same medium, but with 10% FCS.

NH2-Terminal His-Tagged p14
The previously isolated cDNA clone 66b (3) was used as a template for PCR and the PCR fragment corresponding to the p14 sequence was ligated into the plasmid pET-28a(+), Novagen, Madison, WI. The resulting His-tag p14 plasmid was confirmed by sequencing and the expressed His-tagged protein was purified to near homogeneity on a TALON (Clontech, Palo Alto, CA) cobalt-based affinity column and purity confirmed by SDS-PAGE. For studies that did not require the His tag, the tag was removed from the purified protein by thrombin proteolysis using a thrombin cleavage capture kit (Novagen).

p14 Polyclonal Antibody
The above-purified recombinant His-tagged p14 protein was used to generate polyclonal anti-p14 antibodies in rabbits. One milligram of the protein was injected s.c. into rabbits in Freund's complete adjuvant, followed 3 and 6 weeks later by a booster of 1 mg protein in Freund's incomplete adjuvant. The rabbits were bled 3 weeks after the second booster. The serum was used with no further fractionation.

Immunofluorescence
Cells were allowed to attach to a polylysine-coated slide (Sigma, St. Louis, MI) at 37°C for 3 hours in growth medium. The cells were then fixed for 30 minutes in 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS for 3 minutes before incubation with the relevant first and fluorescent-labeled second antibodies. In double-labeling experiments, the two primary antibodies were mixed together. The fluorescence was visualized in a Zeiss Axiovert 200M microscope fitted with a Perkin-Elmer UltraView confocal scanner (Perkin-Elmer, Boston, MA). OpenLab software (Improvision, Coventry, England) was used to image the data.

Immunohistochemistry of Human Paraffin-Embedded Breast Cancer Sections
Commercially available paraffin-embedded sections from 25 different human breast cancer samples and five samples of normal human breast tissue (Chemicon Select Tissue Array, TMA1201-4; Chemicon, Hofhein, Germany) were subjected to immunohistochemical analysis using the polyclonal anti-p14 antibody and the Vectastain ABC kit (Vector, Burlingame, CA) according to a standard protocol supplied by the companies. The immunohistochemical analysis was also confirmed using a DAKO envision plus kit.

Western Blots
Cells were lysed by SDS-PAGE sample buffer, separated by 15% SDS-PAGE and transferred to nitrocellulose. p14 and p21 were visualized using the polyclonal anti-p14 described above, horseradish peroxidase–linked donkey anti-rabbit antibody (The Jackson Laboratory, Bar Harbor, ME) and Pierce Super Signal. The amounts of p14 and p21 were quantitated using a FUJI CCD camera and Image Gauge software.

p14 Binding Proteins
Cell lysate. Logarithmically growing (1 x 109) T-64 cells (see above) were lysed in 1.5 mL hypotonic buffer [20 mmol/L phosphate buffer (pH 7.0), 30 mmol/L NaCl] and sonicated thrice for 20 seconds. The cell lysate was centrifuged for 20 minutes at 16,000 x g, 4°C and the supernatant (cell lysate) was removed and used in the procedures described below.

Cobalt-bound His-tagged p14 affinity column. One milliliter of purified His-tagged p14 (250 µg/mL) in binding buffer [6 mol/L guanidine, 50 mmol/L phosphate buffer (pH 7.0), 250 mmol/L NaCl] was bound to 100 µL Talon (Clontech) cobalt-based affinity column beads as described above. One milliliter of the cell lysate described above was bound to 20 µL of the affinity column by incubating them end over end for 1 hour at room temperature. The beads were then pelleted and extensively washed with 1.4 mL buffer in each of the following steps: (a) twice in 30 mmol/L imidazole, 200 mmol/L NaCl, 40 mmol/L phosphate buffer (pH 8.2), 0.05% Tween 20, 1 mmol/L glycine; twice in 30 mmol/L imidazole, 200 mmol/L NaCl, 40 mmol/L phosphate buffer (pH 7.0); twice in 30 mmol/L imidazole, 200 mmol/L NaCl, 40 mmol/L phosphate buffer (pH 6.5); once in 75 mmol/L NaCl, 20 mmol/L phosphate buffer (pH 7.0). (b) Twenty microliters of SDS-PAGE sample buffer was then added to the washed beads, boiled for 5 minutes and run on a 15% SDS-PAGE gel as described above. The gel was stained with Coomassie brilliant blue and the desired protein bands excised and sent for microsequencing at the Smoler Protein Center, the Technion, Haifa, Israel as previously described (4).

Covalently bound p14 affinity column. A 1 mg aliquot of the thrombinized His-tagged p14 (thmb-p14, see above) was covalently bound to 45 µL CNBr-activated Sepharose 4 FF beads (Amersham, Buckinghamshire, England) under standard conditions as recommended by the manufacturer. Finally, the beads were washed thrice in PBS.

A 1 mL aliquot of the cell lysate described above was bound to 20 µL of the affinity column by incubating them end over end for 1 hour at room temperature. The beads were then pelleted, washed, and the bound proteins removed as described above for the cobalt-bound His-tagged p14 affinity column. In a separate experiment, the washes and elution of this column were varied. The first three sets of two washes were each identical, but the column was then washed once in 500 mmol/L NaCl, 40 mmol/L phosphate buffer (pH 7.0), once in 700 mmol/L NaCl, 40 mmol/L phosphate buffer (pH 7.0), and eluted in 600 µL 1,500 mmol/L NaCl, 40 mmol/L phosphate buffer (pH 7.0). The eluate was concentrated by precipitation in TCA, washed with acetone, dried and resuspended in SDS-PAGE sample buffer, run and analyzed as described above. Both washing methods gave comparable results.

Pulse-Chase Analysis
Exponentially growing cells were centrifuged and washed in low methionine medium. The pellets were then resuspended to a concentration of ~2 x 107 cell/mL in the same medium containing 10% dialyzed FCS and 10 mmol/L HEPES buffer (pH 7.4). The cells were incubated in 450 µL aliquots in a 24-well plate for 30 minutes at 37°C and then [35S]methionine (Amersham SJ90079, 14.3 mCi/mL) was added.

The cells were labeled for 1 hour, at which time methionine was added to 200 µmol/L. One 60 µL aliquot of the cells was removed (time 0). At the end of the indicated times, 60 µL aliquots of the cells were removed into an Eppendorf tube with 1 mL of ice-cold PBS + 2 mmol/L methionine and pelleted at 10,000 rpm for 10 seconds. The pellet was frozen in liquid N2 and stored at –70°C. The frozen cells were defrosted on ice, lysed in 200 µL radioimmunoprecipitation assay buffer [50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1% NP40, 0.5% DOC, 0.1% SDS] and centrifuged for 20 minutes. The supernatants were removed to Eppendorf tubes containing an additional 200 µL radioimmunoprecipitation assay buffer and, after preclearing, incubated 1 hour with 5 µL antibody M-66 at a 1:10 dilution of the hybridoma supernatant. One hundred microliters of protein A-sepharose beads (20%) were then added to each tube and incubated end over end for 2 hours. The beads were washed thrice in 0.5 mL cold TB500SN [10 mmol/L Tris (pH 8.0), 500 mmol/L NaCl, 0.1% NP40], once in 0.5 mL cold TBS and then boiled in 30 µL SDS-PAGE sample buffer for 5 minutes. The immunoprecipitated samples were run on 15% SDS-PAGE, the gels were washed, dried, and radioactivity analyzed using a FUJI phosphorimager.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of polyclonal anti-p14 antibodies. The anti-p14 monoclonal antibody M-66 has been instrumental in our studies (3, 4). However, it only recognizes a single epitope and it does not recognize p14 in cryosections or in paraffin-embedded sections of tumors generated by either T-25 cells or their variants. To overcome these problems, we generated a polyclonal antibody against recombinant p14. This antibody, named {alpha}-p14, specifically recognizes p14 and p21 in both immunofluorescence and Western blot analyses, as well as in paraffin-embedded and cryosections (see below).

p14 in mammary carcinoma. To determine whether the expression and nucleolar localization of p14 is limited to murine lymphomas, or whether it is also present in mammary carcinomas, we have analyzed two murine mammary carcinoma cell lines that contain MMTV, using {alpha}-p14. Both cell lines (4T1 and Mm5MT) express p14, but not p21, as shown on Western blots (Fig. 1B). Immunofluorescence analysis shows that in both mammary carcinoma cell lines (Fig. 2E-H for 4T1 cells and Fig. 2I for Mm5MT cells) p14 is concentrated in the nucleoli. In addition, B23 is colocalized with p14 in Mm5MT cells (Fig. 2J and K), similar to the colocalization in lymphoma cells (4). Thus, import of p14 into the nucleoli is not limited to lymphomas, but seems to be a general property of MMTV-harboring cells.



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  		Figure 2. Nucleolar localization of p14 in mouse mammary carcinoma cells in culture using confocal microscopy. Cells grown on polylysine-coated slides were fixed and permeabilized. p14 was visualized using {alpha}-p14 and fluorescent anti-rabbit second antibodies. A-D, control Rev-2-T-6 lymphoma cells expressing p14; bright field, nuclear staining with 4',6-diamidino-2-phenylindole, p14 staining (green), and overlay of (B) and (C), respectively. E-H, 4T1-mammary carcinoma cell line; bright field, nuclear staining with 4',6-diamidino-2-phenylindole, p14 staining (green), and overlay of (F) and (G), respectively. I-K, Mm5MT mammary carcinoma cell line. I, p14 staining (red); J and K, double staining, p14 (red) + B23 (green).

 
p14 in a subset of human breast cancers. The above findings, taken together with the recent reports on the presence of MMTV-env-related sequences in a subpopulation of human breast cancers (with suggested relation to geographic location; ref. 11) prompted us to determine whether p14 is also expressed in human breast cancers.

Figure 3 compares the translated 5'-env sequence (Genbank accession number AF248270) of the proviral MMTV structure found in the genome of human breast cancer tissues (12), with the p14 amino acid sequence. The two proteins are 86% identical.



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  		Figure 3. Comparison of the sequences of p14 and parallel sequence of the provirus found in a human breast carcinoma (AF248270).

 
As proof of principle, we have analyzed a commercially available tissue array (see Materials and Methods) that contained paraffin-embedded sections from 25 human breast cancer biopsies. This was carried out using polyclonal {alpha}-p14 generated against recombinant p14 (see Materials and Methods). Of the 25 sections probed, 4 (16%) were positive (two strongly positive, Fig. 4D and F; and two others gave weaker signals, not shown). The rest were negative (Fig. 4B), as were five samples of normal human breast tissue supplied on the same slide. Furthermore, nucleoli of the positive tumor cells are stained for p14 (Fig. 5C). Duplicate slides were stained independently at both the Hebrew University and at the NIH with identical findings.



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  		Figure 4. p14 in human breast cancer. A and B, ductal carcinoma; C and D, ductal carcinoma in situ; E and F, invasive ductal carcinoma. A, C, and E, paraffin-embedded sections stained with H&E; B, D, and F, paraffin-embedded sections stained with {alpha}-p14, second antibody coupled to peroxidase and counterstained with hematoxylin. Bar, 100 µm.

 


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  		Figure 5. p14 localizes to the nucleolus in human breast cancer. A-C, invasive ductal carcinoma: H&E staining, p14 staining, and enlarged section from (B), respectively. Bars, 100 and 20 µm for (B) and (C), respectively. Arrows, stained nucleoli.

 
Because p14 is localized in nucleoli from mouse lymphoma, mammary carcinoma, and human breast cancer, it implies that this protein may play a general role, not just in MMTV-associated lymphomagenesis, but also in MMTV-associated mammary carcinogenesis. To start to elucidate the role of p14, we investigated (a) the cellular targets for p14 and (b) whether p14 is a posttranslational processing product of p21.

Cellular targets for p14. One way to investigate the function of p14 is to characterize cellular targets that interact with it. T-64 whole cell lysate was adsorbed onto a Co2+ column to which recombinant, purified His-tagged p14 (see Materials and Methods) was previously bound. Proteins specifically bound to the affinity column were eluted and sequenced (see Materials and Methods; Fig. 6). To gain additional support for the results from the His-tagged p14-Co2+ affinity column, we carried out another set of experiments using a different affinity purification procedure. Here, the His-tag was removed from recombinant p14 by thrombin, and the resultant protein was covalently bound to CNBr-activated Sepharose (see Materials and Methods). This column was then used for affinity purification of p14 binding proteins present in T-64 whole cell lysates, as described above. Only proteins identified by both affinity purification procedures are shown in Table 1. For example, 40S ribosomal protein S4, which has been identified under one set of experiments (see Fig. 6), was not added to Table 1. Because B23 has been previously shown to bind p14 using immunofluorescence colocalization and coimmunoprecipitation (4), its identification here further supports the validity of the present approach. Additional support stems from the finding that, although whole-cell extracts were used for the affinity purification, a substantial portion of the proteins identified as cellular targets for p14 were of nucleolar/nuclear origin (Table 1).



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  		Figure 6. SDS-PAGE of proteins bound to a His-tagged p14 affinity column. Whole-cell lysates of T-64 cells were adsorbed to the affinity column, extensively washed, and eluted with SDS-PAGE sample buffer. A, supernatant of whole-cell extract; B, supernatant of whole-cell extract after absorption onto His-tagged p14 affinity column; C, molecular weight markers; D, eluate of the His-tagged p14 affinity column; E, eluate of control column (without His-tagged p14). Numbers refer to the fractions cut out of the gel and sequenced: (1) NAP-1, similar to tubulin {alpha}1; (2) Etef 1 {alpha}2; (3) autoantigen La; (4) ribosomal protein L5; (5) ribosomal protein L5, nucleolar phosphoprotein B23, 60S acidic ribosomal protein PO, similar to glyceraldehyde-3-phosphate dehydrogenase; (6) ribosomal protein L5; (7) p32-Rack; (8) 40S ribosomal protein S4; (9) ENV MMTV. p14his, NH2-terminal His-tagged p14 initially bound to the cobalt affinity resin and released during elution.

 

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  		Table 1. p14 binding proteins identified through both Co2+- and CNBr-based affinity column purification

 
One of the proteins identified in this way was employed to further evaluate the specificity of the method. p32 was chosen for this, as it is a nonnucleolar protein (15, 16), as antibodies are commercially available, and as it was isolated in each of our ligand fishing experiments. As shown in Fig. 7, p32 was completely depleted from the whole cell lysate by the affinity column, and concentrated in the eluate. The Western blot in this figure is overexposed in order to emphasize the depletion of p32 from the lysate.



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  		Figure 7. p14 affinity column depletes p32 from cell lysates. An extract of T-64 cells was prepared, adsorbed to a His-tagged p14-bound Talon column and the column was washed and eluted as described in Materials and Methods. Samples of the various fractions were run on a 15% SDS-PAGE gel, blotted onto nitrocellulose and visualized with {alpha}-p32 antibodies as described in Materials and Methods. The blot is overexposed to emphasis the amounts of p32 in lanes C-F. A, molecular weight markers; B, eluate of the His-tagged p14 affinity column; C, eluate of the control column; D, T-64 lysate; E, T-64 lysate after adsorption on the control column; F, T-64 lysate after adsorption on the His-tagged p14 affinity column.

 
Pulse-chase experiments. Pulse-chase experiments were conducted as described in Materials and Methods. In pulse experiments, p14 and p21 show similar rates of synthesis, reaching a plateau after 120 minutes (Fig. 8A and B).



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  		Figure 8. p14 and p21 do not share a precursor-product relationship. Pulse-chase analysis of p14 and p21 was carried out on [35S]methionine-labeled, T-64 cells. p14 and p21 were immunoprecipitated and run on SDS-PAGE. The dried gels were analyzed by a FUJI phosphorimager. The individual bands representing p14 and p21 were quantitated using the Image Gauge program. PSL, arbitrary unit of quantitation used by the phosphorimager.

 
For the chase experiments, in order not to saturate the protein pool, cells were labeled for 1 hour with higher levels of [35S]methionine. Again the kinetics of p14 and p21 were similar. p14 showed a t1/2 of 170 minutes, p21 of 214 minutes (Fig. 8C and D). The t1/2 was calculated using a least squares polynomial line fit.

As p14 does not show any lag time of synthesis as compared with p21, nor does p21 degrade prior to p14, the experiments indicate that p21 and p14 do not show a precursor-product relationship, i.e., p14 is not formed principally through proteolytic degradation of p21. Thus, the two proteins may be synthesized either via alternative splicing of the same transcript or through distinct transcripts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have recently shown that the leader peptide of the MMTV Env precursor (p14) is translocated into the nucleoli of murine lymphoma cells harboring this virus (3, 4). Here, we extend these findings to show that mouse mammary carcinoma cell lines containing MMTV, as well as a subset of human breast carcinoma biopsies, also express p14 in their nucleoli. Thus, nucleolar sequestering of p14 may play a role of general significance in growth regulation of MMTV-containing lymphomas and mammary carcinomas.

In addition to the "traditional" role of the nucleolus in rRNA synthesis and ribosome biogenesis, this subnuclear structure has, in recent years, been implicated in a variety of cellular functions such as regulation of the cell cycle, senescence, gene silencing, modification of small nuclear RNAs, and modulation of telomerase function (17–21). A recent study proposed that the nucleolus might also function as a sensor responsive to a wide range of cellular stresses (22). These stresses culminate in the stabilization of p53, a process that, according to these authors, is mediated through disruption of the nucleolus (22). Thus, upon nucleolar disassembly, p14ARF is released from the nucleolus to the nucleoplasm, where it binds to the MDM2 or MDM2-p53 complex, stabilizing p53 by impairing its translocation to the cytoplasm (for a recent review, see ref. 23). An important implication of this model (22) is that functional inactivation of the nucleolus, frequently associated with viral infections (9), will stabilize p53. As p53 is the primary mediator of cellular stress responses, proteins that are bound to p53, either directly or indirectly, may regulate this function. Indeed, the ribosomal protein L5 binds both MDM2 and MDM2-p53, as well as taking part in export of the auxiliary HIV-Rev protein (24). The shuttling protein B23 (nucleophosmin) also interacts directly with both p53 and p14ARF, as well as with HIV-Rev (24). In a recent publication (24), a correlation was found between the expression of mouse mammary tumor-like virus and nuclear accumulation of p53, as well as the presence of progesterone receptor in human female breast cancer.

Based on our findings and the above considerations, we hypothesize that this nuclear accumulation of p53 in human cases of breast cancer is actually mediated through p14. In this respect, it would be of interest to investigate whether those cases of human breast cancer reported in the literature to express MMTV-like sequences, express p14 in their nucleoli. Both B23 and L5 have been identified in this work as cellular targets for p14. Also, we have previously shown (4) that actinomycin D (a stressor which causes nucleolar disruption) causes export of p14 from the nucleolus to the nucleoplasm. We propose that p14 is involved, directly or indirectly, in regulating the stress response. It may also play a role, analogous to that of Rev in HIV-infected cells, in nuclear export or transcriptional regulation. As both p14 and Rev are localized to the nucleolar compartment and bind to similar targets, p14 might be a competitive inhibitor of Rev function in HIV-infected cells. Thus, p14 seems to be a multifaceted protein. However, additional experiments will tell whether it fulfills this promise.

The fact that a substantial number of p14 binding proteins are nucleolar proteins (four of nine), strengthens the validity of our findings, especially as these binding proteins were purified from whole-cell extracts. These findings are also supported by the fact that all p14 binding proteins were assigned this function on the basis of, at least, two independent affinity purification approaches. Further support of these findings comes from our previous work, where B-23 has been shown as a target for p14, based upon colocalization and coimmunoprecipitation analyses (4).

The identification of glyceraldehyde-3-phosphate dehydrogenase and tubulin as binding proteins for p14 is consistent with our preliminary unpublished findings that overexpression of p14 drives T-25-Adh cells—which normally express about half the levels of p14 found in nonadhesive T-25 cells—towards a less adhesive phenotype. Indeed, glyceraldehyde-3-phosphate dehydrogenase has been shown to interact with tubulin in microtubule assembly (25). The interaction of these proteins has also been shown to play a role in modulating the kinetics of membrane trafficking and cellular signaling processes through regulation of membrane fusion that might alter cell adhesion (26).

We also show that p14 can be identified in human breast cancer. This is further supported through nucleolar staining with polyclonal anti-p14. Obviously, a larger number of cases will have to be monitored to determine the true percentage of patients whose cancers express nucleolar p14, and whether this could be correlated with their stage of disease and/or prognosis and/or geographic location. The latter point is of interest, because in a recent study, 42% of Caucasian-Australian women had MMTV-related sequences, whereas <1% of Vietnamese or Vietnamese-Australian women had such sequences (11). Additional support for a human breast carcinoma virus with geographic differences comes from a study in which 74% of Tunisian women with breast carcinoma, compared with 36% of American women, tested positive for MMTV-like sequences (27).

As described above, several independent groups have described the expression of MMTV-related sequences in breast cancer patients (10–13). As the virus can cause both lymphoma and mammary carcinoma in mice, it is of special interest that one group reported the presence of such MMTV-related sequences in patients who have both breast cancer and lymphoma (10). However, this entire issue remains controversial, as other groups suggest, in studies based on gene amplification analysis, that MMTV-related sequences are not present in humans (28–30). We have approached this issue from a different angle, namely the level of expression of a virus-derived protein (p14), as we have the appropriate reagents available for such an analysis. At the protein level, it was claimed as early as the 1970s (31, 32) that MMTV antigens were involved in human breast cancer. These claims, however, have not been substantiated. As p14 has only now surfaced as a possible marker for MMTV-related lymphomas and mammary carcinomas in mice, and in a few cases of human breast cancer, further studies are needed to test the extent of p14 expression in human breast cancer biopsies.

Our recent findings, reported both here and previously (3, 4), of p14 localization to the nucleolus in both mouse and human cancer cells, and its interaction with nucleolar proteins of cellular origin, taken together with the reports of MMTV-related sequences in human breast cancer have provided new impetus for MMTV research and its possible role in the etiology of human breast cancer.


   Acknowledgments
 
Grant support: The Israel Science Foundation, 546/01 and 1308/04 (J. Hochman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: A. Bar-Sinai and N. Bassa contributed equally to this work.

Received 10/31/04. Revised 3/29/05. Accepted 5/27/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Racevskis J. Altered mouse mammary tumor virus transcript synthesis in T-cell lymphoma cells. J Virol 1990;64:4043–50.[Abstract/Free Full Text]
  2. Yanagawa S, Kakimi K, Tanaka H, et al. Mouse mammary tumor virus with rearranged long terminal repeats causes murine lymphomas. J Virol 1993;67:112–8.[Abstract/Free Full Text]
  3. Hoch-Marchaim H, Hasson T, Rorman E, Cohen S, Hochman J. Nucleolar localization of mouse mammary tumor virus proteins in T-cell lymphomas. Virology 1998;242:246–54.[CrossRef][Medline]
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