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Endocrinology Vol. 142, No. 3 1357-1367
Copyright © 2001 by The Endocrine Society

ARTICLES

Identification and Nuclear Localization of a Novel Prolactin and Cytokine-Responsive Carboxypeptidase D1

Catherine K. L. Too2, Nicola Vickaryous, Robert T. M. Boudreau3 and Shirley M. Sangster

Department of Biochemistry and Molecular Biology (C.K.L.T., N.V., R.T.M.B., S.M.S.), Department of Obstetrics and Gynaecology (C.K.L.T.), Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Address all correspondence and requests for reprints to: Catherine K. L. Too, Ph.D., Department of Biochemistry & Molecular Biology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. E-mail: ctoo{at}is.dal.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A full-length, PRL-inducible complementary DNA (cDNA) encoding a novel, nuclear-targeted carboxypeptidase D isoform (designated CPD-N) was identified in the rat PRL-dependent Nb2–11C and PRL-independent Nb2-Sp lymphoma cell lines by differential display. The CPD-N cDNA (3751 bp) has 99% (3582/3583) homology with rat carboxypeptidase D (CPD; 4377 bp). In comparison to the rat CPD cDNA (ORF of 4134 bp; 180-kDa protein), CPD-N was shorter by approximately 600 bases but contained 148 unique bases at the 5'-end to give an ORF of 3399 bp. RT-PCR with primers specific to the 5'-end of CPD-N or to CPD showed that the CPD-N transcript was expressed in the Nb2–11C and Nb2-Sp cells but was not detected in rat brain or lung. Conversely, the CPD transcript was expressed in rat brain but was not detected in the two Nb2 cell lines. CPD-N expression (7.5-kb messenger RNA) was stimulated by PRL (10 ng/ml) and/or by interleukin-2 (24 U/ml) in Nb2–11C and Nb2-Sp cells. Most rat tissues expressed multiple CPD transcripts (7.5, 4.1, and 2 kb). Curiously, CPD transcripts were low or undetectable in male rat liver but readily detected in female liver, suggesting that sex-specific hormone levels may regulate its expression. Indeed, CPD expression in the PRL-responsive HepG2 hepatoma and MCF-7 breast cancer cell lines was low in control cells but was markedly stimulated by PRL after 3 h. Consistent with the shorter ORF of CPD-N, Western analysis detected proteins of smaller molecular sizes of 160 kDa (abundant) and 117 kDa (weak) in the Nb2–11C cells. The Nb2-Sp cells expressed a single and abundant 117-kDa protein, implicating differential protein processing in the two cell lines. Rat CPD has been reported to colocalize with the trans-Golgi network marker TGN38. Subcellular fractionation showed predominant nuclear localization of CPD-N and trace amounts were detected in the 100,000 x g microsomal fraction after PRL treatment (4 h); in contrast, TGN38 was found only in the microsomal fraction at this time. In cells treated with PRL for 24 h, immunofluorescent confocal microscopy showed nuclear and cytoplasmic distribution of CPD-N. Cytoplasmic CPD-N colocalized with TGN-38 whereas nuclear CPD-N had a mesh-like distribution and colocalized with nuclear lamin B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PRL IS INVOLVED IN as many as 85 different biological functions in vertebrates. These actions include osmoregulation, reproduction, growth and development, metabolism, behavior, and immunoregulation (reviewed in Ref. 1). The diverse actions of PRL are mediated by well characterized PRL receptors (PRLR) that are expressed in many tissues including the mammary gland, liver, and lymphoid cells. In addition to the anterior pituitary, PRL is secreted by extrapituitary sites such as the decidua, breast, and T lymphocytes (reviewed in Ref. 2). Therefore, PRL may act as an autocrine-paracrine growth factor as well as an endocrine hormone (1).

The mitogenic action of PRL in immune cells has been studied extensively using the rat PRL-dependent Nb2 lymphoma cell lines (3). Nb2 cells are thymic in origin (4), have specific, high affinity cell surface PRLRs, and are critically dependent on PRL for growth (5). Signal transduction by the PRLR is mediated primarily by the JAK2 tyrosine kinase-STAT (signal transducers and activators of transcription) pathway resulting in the nuclear translocation of the STAT proteins to regulate transcription of PRL-responsive genes (reviewed in Refs. 2, 6). Two other PRLR-associated kinases, serine/threonine kinase Raf1 and tyrosine kinase fyn59, may provide additional signals for lymphocyte proliferation and cell survival. PRLR dimerization and/or Jak2-mediated phosphorylation of the PRLR may activate the MAPK signaling cascade, resulting in the activation of transcription factors like Jun and Fos (2).

In addition to the PRL-dependent Nb2 cell lines, PRL-independent sublines have been established (7, 8). These pre-T Nb2 lymphoma cell lines have provided a useful model for the study of tumor progression of T cell cancers (7, 9). Identification of differentially expressed genes in the PRL-dependent Nb2–11C and PRL-independent Nb2-Sp cell lines may provide insights into the mechanism(s) associated with the emergence of autonomous growth in growth factor- dependent malignant T cell cancers. Using a differential display approach, we have previously identified a number of PRL-inducible genes following acute hormonal stimulation of quiescent Nb2–11C lymphoma cells and some of these genes are constitutively expressed in Nb2-Sp cells (10, 11). In the present study, we describe the cloning and characterization of a novel PRL-inducible carboxypeptidase D (CPD) isoform (designated CPD-N) that is expressed in the Nb2 lymphoma cells. CPD is a recently discovered membrane-bound, B-type metallocarboxypeptidase that is believed to be involved in posttranslational processing of peptides and proteins that transit the secretory pathway (12). We show that the Nb2–11C and Nb2-Sp cell lines express the novel CPD-N, but not CPD, and that expression of CPD-N is stimulated by PRL and/or interleukin-2 (IL-2). PRL also stimulates expression of CPD in the PRL-responsive HepG2 and MCF-7 transformed cell lines. CPD-N is found predominantly in the Nb2–11C cell nucleus where it colocalizes with nuclear lamin B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Suspension cultures of the PRL-dependent rat Nb2–11C lymphoma cells were maintained in Fischer’s medium for leukemic cells supplemented with 100 µM {beta}-mercaptoethanol and containing 10% FBS as a source of lactogens and 10% lactogen-free horse serum (HS) as previously described (13). Confluent Nb2–11C cells (~1.0 x 106 cells/ml) were growth-arrested by culturing cells in medium containing 10% HS alone for 18–24 h. A clonal variant, the PRL-independent Nb2-Sp cell line was maintained in Fischer’s medium containing 10% HS. Both cell lines have doubling times of approximately 20 h and, therefore, before hormonal (mitogen) treatments for up to 24 h, growth-arrested Nb2–11C cells and confluent Nb2-Sp cells were reduced in cell density to about 0.6 x 10 cells/ml with medium containing 10% HS. PRL (10 ng/ml) or IL-2 (24 U/ml) were added for the indicated times, whereas controls (0 h) were left untreated.

Human HepG2 hepatoma and MCF-7 breast cancer cells were cultured in DMEM containing 10% heat-inactivated FBS (56 C for 30 min) supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 µM nonessential amino acids, and penicillin:streptomycin (50 U/ml:50 µg/ml). Before PRL (10 ng/ml) treatment, HepG2 and MCF-7 cells were washed twice with PBS and incubated in DMEM containing 1% heat-inactivated FBS for 24 h.

Differential display, isolation, and identification of PRL-inducible CPD
Differential display of messenger RNA (mRNA) was performed to identify genes which were differentially expressed in the Nb2–11C (± PRL, 3 h) and Nb2-Sp cell lines as previously described (10). A complementary DNA (cDNA) fragment (band 13c.3) was identified in the display gel to be elevated in Nb2–11C cells given PRL for 3 h but not in the untreated controls. Band 13c.3 was constitutively expressed in growing Nb2-Sp cells (10) (see also Fig. 1AGo). Differential expression of band 13c.3 was confirmed with total RNA prepared in a second experiment and by reverse Northern analysis (10). Band 13c.3 was used to screen an Nb2-Sp cDNA library (10) from which was isolated a partial clone, clone-S1. This was cloned into the EcoRI-XhoI cloning site of the pBK-CMV phagemid vector (Stratagene, La Jolla, CA) and sequenced (Sequenase Kit; United States Biochemical Corp., Cleveland, OH); it had 78% nucleotide homology with duck gp180, a glycoprotein belonging to the carboxypeptidase (CP) gene family (14). Clone-S1, initially named CP-Nb2, was used to rescreen the same cDNA library and three additional clones S2, S3, and S4 were isolated. Clone-S1 was also used as a probe in Northern blot analysis. Complete sequencing of clone-S2 was performed by walking primer reactions (Dalhousie University-National Research Council Institute for Marine Biosciences Joint Laboratory, Halifax, Nova Scotia, Canada).



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  		Figure 1. Differential expression of cDNA fragment (band 13c.3) and CP-Nb2. Confluent Nb2–11C cells (Nb2; ~1 x 106 cells/ml) were growth-arrested for 20 h and then reduced to a cell density of 0.6 x 106 cells/ml. The cells were given PRL (10 ng/ml) for 3 h whereas controls were left untreated. Confluent Nb2-Sp cells (Sp) were similarly reduced to a density of 0.6 x 106 cells/ml and harvested at 3 h but without PRL treatment. Total RNA was prepared for (A) differential display of mRNA or (B) Northern analysis as described in Materials and Methods. A, cDNA fragment (band 13c.3) displayed in a 6% sequencing gel was elevated in PRL-treated Nb2 cells (+) but not in untreated controls (-). Band 13c.3 was also elevated in growing Sp cells. Mean ± range from two independent experiments. B, Clone-S1 (CP-Nb2), isolated from a Nb2-Sp cDNA library by band 13c.3, was used as a probe in Northern analysis of Nb2 cells (±PRL for 3 h) as well as of confluent (con) Sp cells or growing Sp cells harvested during this time.

 
Antibodies
Anti-CPD/CPD-N antiserum. Based on nucleotide alignment with duck gp 180, the deduced amino acid sequence of clone-S1 (CP-Nb2) was determined. A synthetic multiantigen peptide (MAP), (HRLRQHHDEYEDEIR)8-MAP, was used to raise rabbit polyclonal antibodies against CP-Nb2 (Research Genetics, Inc., Huntsville, AL) (see Fig. 2Go). CP-Nb2 was later renamed the CPD-N isoform. The antibodies generated were immunoreactive to rat CPD-N and CPD. The preimmune serum and the antiserum were purified using protein A. Briefly, 200 µl of 1 M Tris-HCl, pH 8.0, were added to 1.8 ml aliquots of (anti)serum and the mixture was passed through a protein A-Sepharose CL 4B column preequilibrated with 100 mM Tris, pH 8.0. The column was washed sequentially with 10 volumes of 100 mM Tris, pH 8.0 and 10 volumes of 10 mM Tris, pH 8.0. Antibodies were eluted with Elution Buffer (Pierce Chemical Co., Rockford, IL) and 0.5 ml aliquots were collected for spectrophotometric measurements at 280 nm. The 280 nm protein peak was pooled (~4.0 mg/ml) and stored with 0.2% gelatin-0.1% azide at -20 C.



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  		Figure 2. Schematic diagram comparing rat CPD mRNA with Nb2 CPD clones. The reported rat CPD mRNA has 4377 nucleotides to encode a protein with three CP domains (1 2 3 ), a transmembrane domain (tm), and a cytoplasmic domain (cyto) (12 ). The Nb2 CPD-N clones, S1 to S4, are drawn in relation to the rat CPD mRNA. Clone-S2 is 3751 bp long, its first 148 bases from the 5'-end are unique; the rest of the cDNA has 99% (3582/3583) nucleotide identity with rat CPD. Clone-S2 has an ORF of 3399 bases. > <, CPD-specific primers (415 bp PCR product); bold arrowheads; CPD/CPD-N-specific primers (570-bp PCR product); bold arrows; relative positions of the two primer pairs used to amplify the unique region of 5' CPD-N to give 215-bp and 264-bp products, respectively; the forward 25-mer is common to both sets of primers and its sequence is underlined. Start codon ATG is also underlined. n, Position of deduced amino acids in the cytoplasmic domain used to generate anti-CPD/CPD-N antibodies.

 
Commercial antibodies. Monoclonal anti-trans-Golgi network 38 (TGN38) and anti-PRLR were obtained from Affinity BioReagents, Inc. (Golden, CO). Rabbit polyclonal anti-TFIIB, goat anti-lamin B and horse radish-peroxidase-linked secondary antibodies, donkey antirabbit IgG and goat antimouse IgG, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). AlexaFluor antibodies were from Molecular Probes, Inc. (Eugene, OR)

RNA extraction, Northern analysis and RT-PCR
Total RNAs were extracted from cell lines (Nb2–11C, Nb2-Sp, HepG2, and MCF-7) and rat tissues as previously described (15) or using RNeasy mini kits (QIAGEN, Mississauga, Ontario, Canada). Poly(A)+ RNAs were prepared with Ambion, Inc. (Austin, TX) Poly RNA Kit. Northern blot analysis was performed with multiprime-labeled 32P-CPD-N cDNA (i.e. clone-S1, Fig. 2Go). For RT, the integrity of DNase-treated total RNA was first verified in Northern gels. RT of total RNA (1 µg) was performed in a 25 µl reaction mixture containing M-MuLV reverse transcriptase (100 U; Promega Corp.), 40 pM of random hexamer pd(N)6, 200 µM of deoxynucleotide triphosphates (dNTPs) and 1.6 U RNase inhibitor. The RT reactions were incubated at 23C for 10 min, 42C for 60 min and terminated at 95C for 5 min. A 3-µl aliquot of RT reaction was used for amplification by PCR in a 25 µl reaction mixture containing 200 µM dNTPs, 1.5 mM MgCl2, 2.5 U EnzyONE DNA polymerase (ID Labs Biotechnology, London, Ontario, Canada) and the respective primer pair (25 pmol each/reaction). PCR was performed as follows: 94 C for 75 sec and 25–40 cycles of 94 C for 45 sec, 62–67 C for 45 sec and 72 C for 60 sec; with a final step of 72 C for 5 min. PCR products were electrophoresed in 1 or 1.7% agarose gels. Where possible, primer pairs for PCR were chosen to span one intron-exon splice boundary. For comparative PCR, the linear range of the PCR was determined by varying the number of PCR cycles as previously described (16). The forward and reverse primers used and their respective PCR product sizes (bp) were as follows: CPD/CPD-N, 5'- ATG-GCA-GGG-GTA-TAT-TAA-ATG-CCA-3' and 5'-GGA-TAC-CAG-CAA-CAA-AAC-GAA-TCT-3' (576-bp product for hCPD, 570 bp for rat CPD-N); hPRLR, 5'GGA-CCA-GCA-TCT-AAT-GTC-AGT-3' and 5'-CAC-TTG-CTT-GAT-GTT-GCA-GTG-A-3' (845 bp); hIL-2R, 5'-GTC-ACT-CTA-TAT-GCT-CTG-TAC-AGG-3' and 5'-CCA-CCT-TGT-CTT-CCC-GTG-GGT-CAT-3' (351 bp); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-TGA-TGA-CAT-CAA-GAA-GGT-GGT-GA-3' and 5'-TCC-TTG-GAG-GCC-ATG-TAG-GCC-AT-3' (272 bp). The primer pairs amplifying sequences in the unique 5'-end of CPD-N were 5'-CCT-TTC-TCC-AGC-ACC-AGC-TTT-GCG-3' (forward primer) and 5'-CGG-AGG-TTT-TGC-TAT-AGA-TTC-CAG-TG (reverse primer 1) or 5'-GGG-TGG-TTA-GAA-GCA-TAA-GCT-TTC-3' (reverse primer 2) to give products of 215 bp and 264 bp, respectively. To amplify specifically the 5' end of rat CPD, the forward and reverse primers used were 5'-CGA-GCG-GCT-GGG-ATG-AGC-GGC-CGC-CCT-3' and 5'-GTG-CAT-GTT-ACC-CAC-CAG-CTT-CAC-C-3', respectively, to give a 415-bp product. The 18S primers used were from the QuantumRNA 18S Internal Standards Kit (Ambion, Inc.) and gave a product of 315 bp.

Subcellular fractionation and Western analysis
Nb2–11C cell lysates were used for subcellular fractionation as previously described (11). Briefly, cells (20 x 106 per treatment) were pelleted at 200 x g for 5 min and lysed in cold lysis buffer (500 µl of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA and 50 mM sodium pyrophosphate), containing 3 µg/ml each of aprotinin, leupeptin, pepstatin and 2 mM phenylmethylsulfonyl fluoride for 30 min on ice. The cells were further disrupted by gentle passage through a 25-gauge needle. Whole cell lysates, nuclear (800 x g pellet, 5 min), microsomal (100,000 x g pellet, 60 min) and cytosolic (100,000 x g supernatant, 60 min) fractions were obtained. Protein concentrations were determined and samples were aliquoted for storage at -20 C until further analysis. In three separate experiments, the total protein yields in the nuclear and microsomal fractions were typically 208 ± 34 µg and 217 ± 40 µg, respectively, and 558 ± 98 µg in the cytosolic fractions. SDS-PAGE was performed with 10–20 µg protein/lane that represented 4–5% of total protein in each subcellular fraction. Western blotting was performed with the following primary antibodies: anti-CPD/CPD-N (1:300 of 4 mg/ml IgG), anti-TFIIB (0.5 µg/ml), anti-TGN38 (1:500) and anti-PRLR (1 µg/ml). The horseradish peroxidase conjugated secondary antibodies, donkey antirabbit IgG and goat antimouse IgG, were used at 1:5,000 and 1:1,500, respectively. Immunoreactive signals were detected with Super Signal ULTRA (Pierce Chemical Co., Rockford, IL).

Immunofluorescent confocal microscopy
Nb2–11C cells treated with PRL for 24 h were washed twice with cold PBS. Approximately 70,000 cells (in 100 µl) were cytospun at 91.45 x g for 7 min onto silinated microsope slides, fixed in -20 C acetone for 2 min, and air-dried. The slides were kept at -70 C until further processing. Acetone-fixed cells were treated with 1% paraformaldehyde (wt/vol) in 60 mM L-lysine-0.1 M disodium hydrate orthophosphate, pH 7.4, for 15 min at room temperature. The cells were washed twice in PBS and then permeabilized in PBS containing 0.1% Triton X-100 for 15 min. Cold acetone or methanol fixation are used in some protocols (17, 18) as they provide optimal retention of antigenic determinants and give maximal staining (19). However, it is recognized that acetone or methanol may alter cellular morphology and the localization pattern of some antigens (19). In this study, cells fixed with or without acetone were compared, no difference was observed between the two treatment groups except for a stronger staining in acetone-fixed cells. Detergent-permeabilized cells were washed twice in PBS followed by blocking in 3% BSA-PBS for 1 h at room temperature. In double-labeling experiments, incubation with the first primary antibody (1:10 of 4 mg/ml anti-CPD/CPD-N IgG in 0.1% BSA-PBS) was carried out for 1 h at room temperature, the cells were then washed 3x in PBS and incubated with the first secondary antibody (1:50 of AlexaFluor 488 goat antirabbit IgG conjugate) for 1 h in the dark. All subsequent procedures were performed in dimmed light. The cells were washed 3x in PBS, incubated with the second primary antibody (1:10 of monoclonal anti-TGN38 or 20 µg/ml of goat anti-lamin B) at 4 C overnight, washed again in PBS and then incubated with the second secondary antibody (1:50 of AlexaFluor 594 goat antimouse or AlexaFluor 568 donkey antigoat IgG conjugates) for 1 h at room temperature. After a final 3 washes with PBS, the slides were mounted in Citifluor-glycerol/PBS AF1 solution (Marivac Halifax, Ltd., Halifax, Nova Scotia, Canada). Immunofluorescence was detected by confocal microscopy at 100x magnification.

Statistical analysis
ANOVA and Scheffé’s F test were performed using Statview (Abacus Concepts, Inc., Berkeley, CA, 1992).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning and identification of PRL-inducible CPD-N
By differential display, a gene fragment (band 13c.3) was previously identified to be elevated in PRL-treated Nb2–11C cells but not in untreated controls, and constitutively expressed in Nb2-Sp cells (10) (see also Fig. 1AGo). The PRL-inducible band 13c.3 was extracted from the display gel and used as a probe to isolate from a Nb2-Sp cDNA library clone-S1 (2130 bp). DNA sequencing and initial BLAST analysis indicated that clone-S1 had 78% (725/920) identity with duck Anas sp. gp 180, a glycoprotein that binds hepatitis B virus particles and encoded by a member of the carboxypeptidase (CP) gene family (14). Thus, clone-S1 was initially designated CP-Nb2. Northern analysis confirmed PRL- inducible expression of CP-Nb2 in Nb2–11C cells and constitutive expression in Nb2-Sp cells (Fig. 1BGo).

The deduced CP-Nb2 protein had significant homology with a family of zinc-dependent, regulatory type B-like carboxypeptidases. It had 90% (242/267) amino acid identity with duck Anas sp. gp 180 and 56% (70/125) identity with murine AEBP1, a transcription factor with carboxypeptidase activity (20). By amino acid alignment using BEAUTY (21) and BLASTP (22) searches, CP-Nb2 appeared to have homology with almost the entire length of duck gp180 protein due to three repeated carboxypeptidase domains in gp180 (14). However, CP-Nb2 was clearly distinct from other regulatory carboxypeptidases and was anticipated to be a novel mammalian homolog of the duck carboxypeptidase. From the deduced amino acid sequence of clone-S1, an epitope was identified for the synthesis of a multiantigen peptide, (HRLRQHHDEYEDEIR)8-MAP, to generate antibodies. Clone-S1 was also used as probe to rescreen the Nb2-Sp cDNA library. Three new clones (S2, S3, and S4) were isolated and completely sequenced. BLAST search analysis indicated that these three clones as well as clone-S1 had homology with duck gp 180 as anticipated. However, each of the four clones also had more than 99% nucleotide identity with a very recently reported rat carboxypeptidase D (CPD) (12). The full-length rat CPD was 4377 bases long with an ORF of 4134 nucleotides encoding a 1378-amino acid protein. Like clone-S1, clone-S3 (2094 bp) and clone-S4 (2171 bp) were incomplete (see Fig. 2Go). On the other hand, clone-S2 was a 3751 bp cDNA with an ORF of 3399 nucleotides encoding a predicted 1133-residue protein. Clone-S2 had 99% (3582/3583) nucleotide identity with the reported CPD cDNA. Clone-S2 was shorter by about 600 nucleotides than CPD but had 148 unique bases at its 5' end. Because clone-S2 encoded a unique CPD isoform, it was designated CPD-N (for Nb2 and nuclear, see later) to replace CP-Nb2. The relation of clones S1–S4 to the reported rat CPD cDNA is diagrammatically presented in Fig. 2Go. A comparison of the subunit structure of clone-S2 (CPD-N) with duck gp 180 and CPDs is presented in Fig. 3Go. A comparison is also made to two AEBP1 proteins, each having an active carboxypeptidase domain and shown to bind DNA or to be nuclear-targeted. The mouse AEBP1 is a transcription factor involved in adipocyte differentiation (20), whereas human AEBP1 is believed to play a role in osteoblast differentiation (23).



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  		Figure 3. Comparison of the predicted protein domain organization of clone-S2 (CPD-N) with duck gp 180, CPDs and AEBP1s. Signal peptides, ; carboxypeptidase domains, ; 5'-unique region of CPD-N; ; transmembrane domain, tm; cytoplasmic domain, cyto; nuclear targeting signal (KKPKK), NTS. The mouse AEBP1-DNA binding region ({boxplus}) is in the C-terminus and consists of a region rich in serine, threonine and proline residues (STP-like sequences) flanked by basic and acidic amino acids (H-S Ro, personal communication). The amino acid identities between the carboxypeptidase domains of Nb2 CPD-N, CPDs, and AEBP1s are indicated. aa, Amino acid.

 
We have successfully employed rapid amplification of 5'-cDNA ends (5'-RACE; Marathon cDNA amplification kit, CLONTECH Laboratories, Inc.) to obtain full-length sequences of differential display clones in other studies (manuscripts in preparation). In the present study, 5'RACE was used in attempts to obtain more 5'-end sequences for clone-S2 and/or the 5'-end sequence of CPD from the Nb2-Sp cDNA library. To this end, primers specific either to the unique 148 bases in the 5'-end of clone-S2 or to the 5'-end region common to clone-S2 and the reported rat CPD cDNA were designed. In each case, several different primers were used for 5'RACE but no additional 5'-end sequence was obtained. Therefore, we concluded that only CPD-N was expressed in the Nb2-Sp cells.

PRL and IL-2 stimulate CPD-N expression in Nb2–11C cells
PRL and IL-2 are mitogenic in the Nb2–11C cells that express specific receptors for the two cytokines (24, 25, 26). Northern blot analysis showed that Nb2–11C cells expressed a strong 7.5 kb transcript but a less abundant transcript of approximately 5 kb was also detected. Expression of the 7.5 kb transcript was low, but detectable, in growth-arrested cells and increased markedly by as much as 6- to 8-fold within 4 h of addition of PRL (10 ng/ml) or IL-2 (24 U/ml) (Fig. 4Go).



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  		Figure 4. PRL and IL-2 stimulate expression of CPD in Nb2–11C cells. Growth-arrested Nb2–11C cells were given PRL (10 ng/ml) or IL-2 (24 U/ml) and the cells were harvested for total RNA extraction at the indicated times. Northern analysis of CPD was performed using 32P-labeled clone-S1. Ethidium bromide-stained 28S ribosomal RNA (rRNA) was used to verify RNA loading and to standardize relative expression. The latter was determined by densitometric scanning (lower panels). Representative of three separate experiments, each demonstrating stimulation of CPD-N expression by PRL or IL-2.

 
Figure 1Go shows that CPD-N was expressed constitutively in PRL-independent Nb2-Sp cells (no hormone treatment). However, Nb2-Sp cells have PRL receptors (27) and we tested by RT-PCR the effect of PRL on CPD-N expression in these cells (see Fig. 2Go for primers used). PRL treatment further elevated CPD-N expression by 3- to 4-fold in the Nb2-Sp cells (Fig. 5Go).



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  		Figure 5. PRL stimulates CPD expression in Nb2-Sp cells. Confluent Nb2-Sp cells (1 x 106 cells/ml) were reduced in cell density (0.6 x 106 cells/ml) as in Fig. 1Go. The cells were treated with PRL (10 ng/ml) for the indicated times. RT-PCR was performed with CPD-specific (see Fig. 2Go) and 18S rRNA-specific primers to give predicted PCR products of 570 bp and 315 bp, respectively. PCR with each primer set was in the linear range (CPD, 30 PCR cycles; 18S, 25 cycles). M, 100-bp DNA markers. Representative of two independent experiments, each showing stimulation of CPD-N expression by PRL.

 
CPD expression is stimulated by PRL in HepG2 and MCF-7 cells
CPD transcripts of approximately 8 and 4 kb, and additional transcripts of 2–5 kb have been reported in most rat tissues examined (12). We have also shown by Northern analysis multiple CPD transcripts of 7.5, 4.1, and/or 2 kb in rat brain, testis, lung, and heart (Fig. 6AGo). Because CPD-N expression was stimulated by PRL in the Nb2–11C cells, CPD expression in two normal PRL target tissues, the liver and mammary glands, was investigated. Lactating female rats (with physiologically elevated levels of circulating PRL) vs. virgin female rats were used and compared with male rats. Northern analysis using poly(A)+ RNA (2 µg/lane) detected CPD transcripts of 7.5 kb and 4.1 kb in livers of female virgin and lactating rats, but CPD expression was very low or undetectable in the male rat liver (Fig. 6BGo). No CPD transcript was detected in the mammary glands of lactating rats (Fig. 6BGo). Poly(A)+ RNA yields were low from the limited amount of mammary gland tissues from virgin rats, and no transcript was detected (data not shown).



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  		Figure 6. CPD is widely expressed in rat tissues and is stimulated by PRL in cancer cells. A, Northern analysis was performed with total RNA (25 µg/lane) prepared from male rat tissues. B, Northern analysis was performed with poly(A)+ RNA (2 µg/lane) from male and female (virgin or lactating) rats; mam. gld., mammary glands. Representative of two independent RNA extractions from three to four male or female rats. C, HepG2 and MCF-7 cells were cultured in 1% heat-inactivated FBS for 24 h, as described in Materials and Methods, before PRL (10 ng/ml) treatment for 3 h. Total RNA (1 µg/sample) was isolated for RT-PCR. M, 100-bp DNA markers. The sizes of the PCR products are shown on the right.

 
Because CPD was more strongly expressed in female than in male rat liver and was undetectable in rat mammary glands, CPD expression in transformed liver (HepG2) and mammary (MCF-7) cell lines was examined. HepG2 and MCF-7 cells, routinely maintained in 15% heat-inactivated FBS, were cultured in 1% of FBS for 24 h before PRL (10 ng/ml) treatment for 3 h. Using hCPD-specific primers, a weak PCR product (576 bp) was detected in control HepG2 cells but not in control MCF-7 cells. However, hCPD expression in both cell lines was markedly increased with PRL treatment (Fig. 6CGo). The two cell lines were also confirmed to express the hPRL receptor (Fig. 6CGo).

Expression of CPD-N is specific to Nb2 cells
Northern analysis showed expression of CPD in rat brain and lung (Fig. 6AGo). To investigate whether the CPD-N isoform was also expressed in these tissues, RT-PCR was performed with two sets of primers that would amplify sequences unique to the 5' end of CPD-N (see Fig. 2Go). The predicted PCR products of 264 bp and 215 bp, respectively, were detected in Nb2-Sp and Nb2–11C cells but not in rat brain or lungs, even with up to 40 cycles of PCR (Fig. 7Go). Conversely, to determine whether CPD was expressed in the Nb2 cells, PCR was performed with primers specific to the 5' end of rat CPD. A predicted product of 415 bp was detected in rat brain but not in rat lung nor in the Nb2 cells (Fig. 7Go). The CPD-specific primers used were based on the reported rat CPD sequence, the 5' end of which was determined from different PCR clones of rat brain and phaechromocytoma PC12 cells (12). Therefore, the presence of a transcript in rat brain but not in rat lungs may be due to nucleotide differences in neural and lung tissue. Nonetheless, these results show that rat Nb2 lymphoma cells express CPD-N, whereas the CPD transcript was undetectable or absent in these cells. The CPD-N transcript was not detected in either of the two rat tissues tested.



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  		Figure 7. CPD-N expression is Nb2 cell specific. Total RNAs (1 µg/sample) from Nb2-Sp (Sp), Nb2–11C (Nb2) cells, rat brain, or lungs were isolated for RT-PCR. PCR was performed with two primer pairs specific to the unique 5'end of CPD-N to give PCR products of 264 or 215 bp (see Materials and Methods and Fig. 2Go) or using a primer pair specific to the 5' end of rat CPD to give a 415-bp product (35–40 cycles of PCR). The 18S rRNA product showed equal amounts of RNA (25 cycles and in linear range of reaction). Negative controls (no RT step) gave no product (data not shown). M, 100 bp DNA markers. Representative of three experiments.

 
Western analysis and subcellular localization of CPD-N
The reported rat CPD cDNA encodes a 1378-residue glycoprotein of 180 kDa (12). A smaller protein was expected from the 1133-residue CPD-N in the Nb2 cells. The antibodies used in the present study were raised to a region in the cytoplasmic domain that was identical for CPD-N and rat CPD (see Fig. 2Go). Western analysis showed a strong immunoreactive band of 160 kDa and a faint band of 117 kDa in Nb2–11C cells (Fig. 8AGo). A single, strong band of 117 kDa was detected in the Nb2-Sp cells. Multiple immunoreactive bands of 210, 160, 140, and 120 kDa were detected in the rat brain. Bands of approximately 180, 163, 145, and 117 kDa were seen in rat lung. The preimmune serum detected nonspecific bands of 95 and 130 kDa (Fig. 8AGo, arrowed) which may or may not be detected by the antiserum.



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  		Figure 8. Western analysis of CPD protein. A, Whole cell homogenates of Nb2–11C (Nb2) cells (+ PRL for 30 min), Nb2-Sp (Sp) cells, rat brain, and lung were used for Western analysis with anti-CPD-N/CPD antiserum or preimmune serum as described in Materials and Methods. Cell lines, 20 µg protein/lane; tissues, 10 µg protein/lane, representative of two experiments. B, Growth-arrested Nb2–11C cells (0 h) were given PRL (10 ng/ml) for 1 and 4 h. Nuclear (nuc; 800 x g), microsomal (mic; 100,000 x g pellet) and cytosolic (cyt; 100,000 x g supernatant) fractions were used for Western analysis (10–20 µg protein/lane, representing ~4% of total protein in each fraction). Representative of three independent experiments. C, Densitometric analysis of nuclear CPD-N. *, P < 0.05 (n = 3).

 
The 180-kDa CPD protein is primarily distributed on intracellular membranes, particularly in the Golgi, but soluble forms are also present (28, 29, 30). CPD has also been shown to colocalize with furin and TGN38, markers for the trans-Golgi network (31, 32). To examine the subcellular distribution of CPD-N, Nb2–11C cell lysates were further fractionated. The 160-kDa CPD-N was found predominantly in Nb2–11C nuclear fractions, but trace amounts were detected in the microsomes after addition of PRL (Fig. 8BGo). PRL treatment for 4 h significantly (P < 0.05) increased the nuclear levels of CPD-N (Fig. 8CGo). The purity of the subcellular fractions was confirmed by the exclusive detection of TFIIB (36 kDa) and TGN38 (75–80 kDa), the integral membrane protein of the trans-Golgi network, in the nuclear fraction and membrane fractions, respectively. The PRL receptor in Nb2–11C cells was detected as doublet bands (58 and 62 kDa); it was abundant in the cytosol but was also present in the membrane fraction (Fig. 8BGo). Thus, in growth-arrested Nb2–11C cells or cells given PRL for 4 h, CPD-N was predominantly in the cell nucleus, whereas TGN38 was in the membrane fraction.

The monoclonal anti-TGN38 antibody used was described by the supplier to be rat specific and was predicted to detect a 38- to 41-kDa immunoreactive band. The detection of 75–80 kDa TGN38 in the Nb2–11C cells is not unusual. The TGN38 protein may be highly glycosylated or anomalously folded and has been reported to give an apparent Mr of 85–95 on SDS/PAGE (18, 33).

Localization of CPD-N by immunofluorescent microscopy
Nb2–11C lymphoma cells have relatively large nuclei. The cytoplasm appears greatly diminished in growth-arrested cells but is restored following 24 h of PRL treatment (data not shown). To further examine subcellular distribution of CPD-N in relation to TGN38, Nb2–11C cells were treated with PRL for 24 h to give the cells a distinguishable cytoplasm and also to induce CPD-N protein expression. The Nb2–11C cells have a doubling time of approximately 20 h after PRL stimulation (34), and, therefore, are also in the mitotic phase at this time. Double-fluorescent labeling experiments with anti-CPD-N and anti-TGN38 were performed followed by confocal microscopy (Fig. 9Go, A–C). CPD-N was detected in the cytoplasm and in the cell nucleus where it appeared to have a mesh-like distribution (Fig. 9AGo; see also 9D). As expected, the trans-Golgi network marker TGN38 was localized predominantly in the cytoplasmic compartment (i.e. in the trans-Golgi membranes), but some nuclear staining was detected (Fig. 9BGo). In the original characterization of TGN38, immunofluorescent analysis of cold methanol-fixed cells showed predominant localization of TGN38 in the trans-Golgi network, but also with some nuclear staining (18). Double-immunofluorescent labeling showed strong colocalization of CPD-N with cytoplasmic TGN38 in these cells (24 h PRL), indicating that CPD-N was associated with or in the trans-Golgi network. This is consistent with the detection of CPD-N in the microsomal fractions of PRL (4 h)-treated cells (Fig. 8BGo). More significantly, CPD-N had a distinct distribution in the cell nucleus (Fig. 9CGo). Distinct nuclear localization of CPD-N was also seen in growth-arrested cells, but the thin cytoplasm of these cells made visualization of cytoplasmic staining difficult (data not shown).



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  		Figure 9. Immunofluorescent detection of CPD-N, TGN38 and nuclear lamin by confocal microscopy. A–F, Nb2–11C cells were treated with PRL (10 ng/ml) for 24 h and prepared for confocal microscopy. Double-fluorescent labeling was sequentially performed with the first set of primary antibodies and its AlexaFluor-conjugated secondary antibodies and then followed by the second set of primary/secondary antibodies as described in Materials and Methods. A, Rabbit anti-CPD-N/AlexaFluor 488 goat antirabbit IgG; B, monoclonal anti-TGN38/AlexaFluor 594 goat antimouse IgG; C, composite of A and B. D, Rabbit anti-CPD-N/AlexaFluor 488 goat antirabbit IgG; E, goat anti-lamin B/AlexaFluor 568 donkey antigoat IgG; F, composite of D and E. Bar, 10 µm.

 
The nuclear, mesh-like distribution of CPD-N prompted us to investigate the possibility of CPD-N association with nuclear lamins. Lamins (A, B, and C) form a fibrous proteinaceous meshwork underlying the inner nuclear membrane but may also be localized in the nuclear interior in a diffuse or spotted pattern (35). Lamins also shows enhanced localization in the cytosol of mitotic cells (36). Cancer cell lines, including the Raji lymphoblastic cell line, consistently express lamin B, whereas lamins A and C may be absent (37, 38). In the present study, double-immunofluorescent labeling for CPD-N and lamin B (Fig. 9Go, D–F) showed the same pattern of distribution for CPD-N as described above (see Fig. 9Go, A and D). Lamin B was detected in the nucleus of growth-arrested cells (data not shown), but was localized in the cell nucleus as well as in the cytoplasm of PRL-treated (24 h) cells (Fig. 9EGo). Double-labeling showed colocalization of CPD-N with lamin B in both nuclear and cytoplasmic compartments (Fig. 9FGo).

hCPD is expressed in human immune cells and cancers
Because Nb2 cells are T-lymphoma cells, we examined CPD expression in human immune cells and lymphoid cancer cell lines. Northern analysis of commercial blots showed multiple transcripts of hCPD (strong 10.3, 8.9, and 7.4 kb transcripts and a weak 5.6 kb transcript) in human immune tissues (Fig. 10AGo). With the exception of Burkitt’s lymphoma, these hCPD transcripts were also detected in a variety of human tumor cell lines such as promyelocytic leukemia HL-60, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, as well as HeLa S3, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361 (Fig. 10BGo).



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  		Figure 10. Multiple transcripts of hCPD are expressed in human immune tissues and cancer cell lines. Northern analysis of hCPD expression in (A) 1, spleen; 2, lymph node; 3, thymus; 4, lymphocytes; 5, bone marrow and 6, fetal liver and, (B) 1, promyelocytic leukemia HL-60; 2, HeLa cell S3; 3, chronic myelogenous leukemia K-562; 4, lymphoblastic leukemia MOLT-4; 5, Raji Burkitt’s lymphoma; 6, colorectal adenocarcinoma SW480; 7, lung carcinoma A549 and 8, melanoma G361. GAPDH was used to verify presence of poly(A)+ RNA on the commercial blots. (C) hCPD is expressed in human lymphomas. Total RNA from human lymphomas were used for RT-PCR using specific primers as described in Materials and Methods. Predicted PCR product sizes are on the right. GAPDH served as a positive control for the RT-PCR.

 
CPD expression was also examined in clinical samples of human lymphomas by RT-PCR. Some of these samples expressed receptors for IL-2 and/or PRL. Five of the eleven samples tested strongly expressed hCPD (samples 1, 4, 5, 10, and 11), whereas sample 9 expressed low levels of the hCPD transcript (Fig. 10CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified a novel PRL- and IL-2-inducible full-length cDNA (clone-S2; 3751 bp) that encodes an isoform of rat CPD. In comparison to rat CPD, the 5' end of clone-S2 is shorter by about 600 nucleotides but is replaced by 148 unique bases. Clone-S2 has an ORF of 3399 bases encoding a 1133-residue protein that we have designated CPD-N (for Nb2 and nuclear). Rapid amplification by 5'-RACE of the Nb2-Sp cDNA library using primers specific to the unique 5'-end sequence of clone-S2, or using primers specific to the 5'-end sequence common to clone-S2 and the rat CPD cDNA, did not yield any additional product. Therefore, we concluded that CPD-N, but not CPD, is expressed in the rat Nb2 lymphoma cells.

CPD is the mammalian homolog of duck gp180 and is a B-type metallocarboxypeptidase (14, 29). Unlike other members of the carboxypeptidase gene family with molecular mass of 30–40 kDa (e.g. the digestive CPA and CPB) or 50–60 kDa (e.g. the peptide-processing CPE and cpm), CPD is a 180-kDa protein. The large size of CPD is due to three repeats of a 50-kD CPE-like metallocarboxypeptidase domain, the latter is believed to arise from tandem duplication of an ancestral carboxypeptidase-coding sequence (14). The rat (12), human, and mouse (39) CPD cDNAs have been cloned. The nucleotide sequence of the full-length rat CPD cDNA was determined from a number of clones isolated from cDNA libraries of rat testis and hippocampus or from clones produced by PCR using RNA from rat brain and pheochromocytoma PC12 cells or from a clone deposited in GenBank (12). The deduced rat CPD protein has several motifs in CP-domain 1 including three Zn2+-binding residues, three substrate binding/active site residues, and an integrin-binding sequence (RGD) found in many peptide-processing endopeptidases (40). Rat CPD is a glycoprotein with 15 potential Asn glycosylation sites (12). Multiple transcripts of rat CPD have been detected in most tissues examined, consistent with a proposed role in the posttranslational processing of peptides and proteins that transit the secretory pathway (see review in Ref. 28).

The novel Nb2 CPD-N cDNA has a shorter ORF than rat CPD cDNA, but it is a full-length clone. The deduced CPD-N protein has no a signal peptide nor integrin-binding sequence. The CP-domain 1 of CPD-N is truncated but it has one Zn2+-binding residue, two substrate binding/active site residues, and 13 potential Asn glycosylation sites. CPD-N has two intact CP-domains and is anticipated to be enzymatically active. The CPD-N ORF predicts a smaller, 126-kDa protein, but our antibodies specific to the cytoplasmic domains of CPD-N or CPD, detected a predominant immunoreactive band of 160 kDa and a less abundant band of 117 kDa in the Nb2–11C cells. Only the 117-kDa immunoreactive band is expressed abundantly in the Nb2-Sp cells, suggesting that the CPD-N protein is processed differently (i.e. posttranslational glycosylation and/or N terminus proteolytic cleavage) in the two cell lines. Rat CPD itself is predominantly 180 kDa (12) but molecular mass of 140, 115, and 100 kDa have also been reported in various rat tissues, the smaller forms of CPD may contain different C-termini (30). We showed that rat brain and lung homogenates have multiple immunoreactive bands ranging from 117 to 210 kDa. A 180-kDa band was present in rat lung but not in the brain, the latter has a 210-kDa immunoreactive band. Thus, rat brain CPD may exist as a larger protein (210 kDa), which undergoes proteolytic cleavage to give the 180-kDa protein. More importantly, neither 180 kDa nor 210 kDa CPD-specific bands were detected in the Nb2 lymphoma cell lines, further evidence that CPD-N (160 kDa), but not CPD, is expressed in these cells.

The third evidence supporting expression of CPD-N, but not CPD, in the Nb2 cells was provided by RT-PCR using primers specific to the 5'-end of CPD-N or to the 5'-end of CPD. Rat brain and lung were used as controls as they express CPD transcripts ( (12) and present study). The 5'-end CPD-N-specific primers gave predicted PCR products (215 bp or 264 bp) in Nb2–11C and Nb2-Sp cells but not in rat brain or lung. Conversely, the primers specific to the 5'-end of CPD gave the predicted 415-bp PCR product in rat brain but not in rat lungs nor in the Nb2 cells. The presence of a 5'CPD-specific transcript in rat brain but not in rat lung may be due to nucleotide differences in rat neural vs. lung tissues.

Major CPD transcripts of 4 and 8 kb are widely expressed in rat and human tissues (12, 39, 41). Additional transcripts from 1.4 to 5 kb are also present in some tissues or cell lines (12). Despite the inability to detect CPD mRNA in rat liver (12), Western analysis has detected soluble and membrane forms of CPD in this tissue as well as in rat brain, heart, and kidney (30). By Northern analysis, we showed a wide distribution of CPD transcripts (7.5, 4.1, and 2 kb) in most normal male rat tissues. Curiously, CPD transcripts (7.5 and 4.1) were barely detectable in the male rat liver, but readily detected in female rat liver, suggesting that circulating sex-specific hormone levels may stimulate CPD expression. CPD has been shown to be strongly expressed in HepG2 hepatoma cell cultures (41). HepG2 and MCF-7 breast cancer cell lines are known PRL target tissues, and we confirmed their expression of the hPRL receptor. By reducing the FBS concentration to 1% before PRL treatment, we showed that hCPD expression was weak or undetectable in control HepG2 or MCF-7 cells but was markedly stimulated by PRL. Therefore, like PRL-stimulation of CPD-N in the rat Nb2 lymphoma cells, PRL also stimulates hCPD expression in HepG2 and MCF-7 cells.

A role for CPD in the processing of proteins that transit the secretory pathway was suggested when it was colocalized with the trans-Golgi network markers, furin and TGN38 (31, 32). Surprisingly, we showed by subcellular fractionation predominant localization of CPD-N in the nuclear fractions of Nb2–11C cells. PRL treatment for 4 h significantly increased nuclear CPD-N protein levels and, furthermore, trace amounts of CPD-N appeared in the microsomal fractions. The TGN38 protein was found exclusively in the microsomal fractions during this period. In cells treated with PRL for 24 h, confocal microscopy showed strong nuclear and cytoplasmic distribution of CPD-N. The cytoplasmic CPD-N colocalized in part with TGN38, suggesting increased protein synthesis and/or trafficking into the trans-Golgi network. CPD-N lacks a N-terminal signal peptide (see Fig. 3Go) but may have some other as yet unidentified motif(s) that targets it to the trans-Golgi network. For example, TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain (42, 43). The cycling of furin from the plasma membrane to its predominant intracellular localization in the trans-Golgi network is determined by three endocytosis motifs in its cytoplasmic domain (44, 45). A C-terminal di-leucine targets the Menkes protein from the plasma membrane to the trans-Golgi network (46). Confocal microcopy also showed colocalization of nuclear and cytoplasmic CPD-N with lamin B. As indicated earlier, lamins are nuclear membrane proteins (35). However, lamins undergo reversible mitotic disassembly (47, 48) and show enhanced localization in the cytosol of mitotic cells (36). Nuclear localization of CPD-N is unusual but not unique. The adipocyte-enhancer binding protein (AEBP) 1 is a transcriptional repressor with carboxypeptidase activity (20). AEBP1 has one regulatory B-like carboxypeptidase domain and a C-terminus DNA-binding domain (see Fig. 3Go) that binds the adipocyte enhancer 1 sequence of the adipose P2 (aP2) gene. AEBP1-DNA interaction enhances its protease activity and represses aP2 gene expression (49).

The role of the TGN- and nuclear-localized CPD-N in the Nb2 lymphoma cells is as yet unknown. Whether CPD-N is also expressed in other PRL-responsive tissues remains to be examined. The significance of CPD-N colocalization with lamin B is also not known. Lamins have been implicated in cellular processes including DNA replication, chromatin organization, and nuclear growth. The structural and functional integrity of the nuclear lamina during the cell cycle is regulated by covalent modifications, such as prenylation and carboxymethylation, as well as by proteolytic cleavage, for the correct assembly of newly synthesized lamins at interphase (48, 50, 51). The nuclear lamina also undergo reversible disassembly during mitosis (47, 48). Whether CPD-N is involved in this process in the Nb2 lymphoma cells remains to be seen. The enzymatic properties and functional role of CPD-N in Nb2 lymphoma cells are under further investigation.


   Acknowledgments
 
We are grateful to Dr. Geoffrey Rowden (Department of Pathology) for excellent advice on immunofluorescent microscopy and discussions on the nuclear lamins. We would like to thank Dr. Annette Foyle (Department of Pathology) for the human lymphoma samples and Dr. Michael Wilkinson (Department of Obstetrics and Gynaecology) for female rat poly (A)+ RNAs. The assistance of Ms. Patricia Colp (Pathology) and Mr. Steven Whitefield (Anatomy) in immunofluorescent and confocal microscopy are gratefully acknowledged. It is a pleasure to thank Dr. Paul R. Murphy for critical discussions.


   Footnotes
 
1 This study was supported by MRC Grant MOP-12895 (to C.K.L.T.). The GenBank accession number for CPD-N (clone-S2) is AF284830. Back

2 Scholar of the Medical Research Council of Canada (MRC). Back

3 Supported by a Cancer Research and Education (CaRE)-Nova Scotia trainee award, with funding from the Faculty of Medicine, Dalhousie University. Back

Received July 11, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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M. R. de la Vega, R. G. Sevilla, A. Hermoso, J. Lorenzo, S. Tanco, A. Diez, L. D. Fricker, J. M. Bautista, and F. X. Aviles
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