| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
1 College of Pharmacy and 2 Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio, and 3 Department of Comparative Biosciences, University of Wisconsin, Madison, Wisconsin
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In addition to the original Nb2 lymphoma cell line, the Nb2-SFJCD1 subline was developed from parental cells by prolonged lactogen starvation and cloning of the surviving cells. This subline is PRL independent for proliferation (2) and is highly metastatic when transplanted s.c. into Noble rats in contrast to the parental line (3) . By exploiting the differences as well as common features of these Nb2 cell lines, we have used this model to investigate molecular mechanisms that may underlie tumor progression in hormone-dependent cancers.
Breast cancer, which is frequently hormone responsive, is the most common form of cancer and is the second leading cause of death in women (4
, 5)
. The etiology of this disease most likely reflects contributions by numerous factors, including menstrual and reproductive history, genetic predisposition, prolonged estrogen therapy, dietary factors, and previous atypical benign breast disease (6, 7, 8)
. The majority of human breast cancers develop as hormone-responsive tumors. As a result, 
60% of those that express estrogen receptors respond to antiestrogen or other endocrine therapies (9
, 10)
. However, with malignant progression, breast cancers frequently progress to a more virulent phenotype, characterized by hormone-independent growth, resistance to chemotherapy, and increased invasiveness that can result in widespread, life-threatening metastases (11
, 12)
. The contribution of PRL to breast cancer has been controversial. However, reports of local synthesis of PRL by mammary epithelial cells may explain much of the inconsistent data. The relationship of PRL to expression of receptors for ovarian hormones in other reproductive tissues, as well as many breast tumors, underscores the importance of the study of this hormone (13, 14, 15)
. A long-term goal of our laboratory is to identify new targets for pharmacotherapy of breast cancer. Thus, we are investigating genes that are regulated by hormones in normal mammary epithelium that may be dysregulated in breast tumors.
Using mRNA differential display in the Nb2 cell model, we searched for novel hormone-dependent genes in Nb2-11 cells and those that may be expressed in exponentially proliferating and differentiating agent [sodium butyrate (NaBT)]-treated Nb2-SFJCD1 cultures. Initially, numerous differentially expressed transcripts were identified. DNA sequencing and GenBank analysis of the cloned 3'-cDNAs yielded a 289-bp fragment that appeared to be novel. Herein, we report the cloning and characterization of a previously undescribed, hormone-regulated, proliferation-associated cDNA (HRPAP20; GenBank accession no. NM_198783), the expression of which is regulated by hormones and differentiating agents in rat Nb2 lymphomas and in human breast adenocarcinoma cells. Its constitutive expression increased proliferation and antagonized apoptosis in hormone-dependent tumor cells.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Protein kinase inhibitors H7, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine·2 HCl [protein kinases A, G, and C (PKA, PKA, and PKC, respectively)]; H8, N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide·2 HCl (PKA); and RO 31-8220, 2-[1-(3-(amidinothio)propyl)-1H-indol-3-yl]-3-(1-methylindol-3-yl) maleimide·methanesulfonate (PKC) were obtained from Biomol.
Cell Culture.
The PRL-dependent, cloned rat pre-T lymphoma cell lines Nb2-11 and Nb2-U17 and the PRL-independent subline, Nb2-SFJCD1, were obtained from Peter W. Gout (British Columbia Cancer Agency, Vancouver, British Columbia, Canada). Nb2-11 cell cultures were maintained at 37°C in Fischer’s medium containing 10% fetal bovine serum (BioWhittaker, Walkersville, MD) as a source of lactogen, 10% horse serum (BioWhittaker), 2-mercaptoethanol (10-4 M), penicillin (50 units/ml), and streptomycin (50 mg/ml; maintenance medium), as originally described for the parental Nb2 cell line (3)
. The Nb2-SFJCD1 subline was cultured in maintenance medium from which fetal bovine serum was omitted. In some experiments, Nb2-11 cells were rendered quiescent by preincubation for 18–24 h in PRL (lactogen)-free medium, i.e., Fischer’s medium supplemented with 2-mercaptoethanol, antibiotics, and 10% nonmitogenic gelding serum (ICN, Irvine, CA). Under these conditions, Nb2-11 cells were arrested in the early G1 phase of the cell cycle, whereas Nb2-SFJCD1 cells continued to proliferate. Proliferation of Nb2-SFJCD1 cells was arrested by pretreatment with differentiating agents, including NaBT (2 mM, 72 h), all trans-RA (10 µM, 72 h), or vitamin D3 (100 nM, 24 h). Before each experiment, differentiating agents were removed by washing; the cells were subsequently resuspended in lactogen-free medium before the addition of PRL.
Human breast carcinoma MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM containing 10% fetal bovine serum and antibiotics. Before experimentation, the medium was replaced with phenol red-free DMEM containing 1% insulin, transferrin, and selenium (Collaborative Biomedical Products, Bedford, MA), and the cells were cultured for 24 h.
Stable PRL-deficient MCF-7 cells were generated as described by Schuler et al. (16)
by transfecting MCF-7 cells with the MinTK (thymidine kinase) or GAS3TK (IFN-
-activated sequence) vector (13)
. Stable transfectants containing the MinTK or GAS3TK vector were selected in 50 µM ganciclovir and screened for PRL mRNA. Clones of PRL-deficient MCF-7 cells were passaged continuously in the presence of ganciclovir and remained PRL responsive for 20 passages. The cells were cultured in serum-free RPMI 1640 for 48 h, then treated with PRL (4 nM) for 12 h before immunoblot analysis.
mRNA Differential Display.
Total RNA obtained from PRL-treated and control Nb2-11 and NaBT-treated Nb2-SFJCD1 cells were subjected to differential mRNA display using a RNA map kit (GenHunter, Nashville, TN). After reverse transcription with murine Moloney leukemia virus reverse transcriptase, an aliquot of the reaction was used for PCR amplification using an oligo-dT primer (T12MN) and arbitrary primers in the presence of deoxynucleotide triphosphates containing 35S-dATP (specific activity 1250 Ci/mmol; New England Nuclear, Boston, MA) and Taq polymerase (Life Technologies, Inc., Grand Island, NY). PCR was conducted at 92°C for 30 s, 40°C for 30 s, and 72°C for 30 s for 40 cycles. The resulting reaction products were resolved on 6% denaturing polyacrylamide gels. After exposure to X-ray film, the differentially expressed bands were identified, isolated, and sequenced (University of Cincinnati DNA Core Facility). An apparently novel 289-bp 3'-sequence was identified; it was used as a probe for Northern blot analysis of Nb2-11, Nb2-SFJCD1, and MCF-7 cells.
cDNA Cloning and Cell Transfection.
Total RNA was prepared from rat Nb2 cell pellets as described previously (17)
. Amplification of the target cDNA was accomplished using rapid amplification of complementary ends (RACE)-PCR using a Touchdown PCR protocol (Clontech, Palo Alto, CA). The annealing temperature during the initial rounds of RACE-PCR was held at the Tm of the specific primers to facilitate gene-specific synthesis. During subsequent PCR rounds, the annealing temperature was lowered to the Tm of the universal primer. Using total RNA, first-strand synthesis was conducted according to the manufacturer’s recommendations (Clontech) using 3'-RACE cDNA [5'-AAGCAGTGGTAACAACGCAGAGTAC(T)30 N-1N-3'; n = A, C, G, or T; N-1 = A, G, or C], 5'-RACE cDNA synthesis primers 5'-(T)25N-1N-3'; n = A, C, G, or T; N-1 = A, G, or C, and murine Moloney leukemia virus reverse transcriptase. First-strand cDNAs were used in the 5'- and 3'-RACE-PCR reactions. Two gene specific primers were synthesized 5'-CGGTCGCTTCTCCCCTCTACTTACC-3' and 5'-CATGACTGAGCACAAAGGATCCACCA-3' (University of Cincinnati DNA Core Facility). These were designed to have >50% GC content and Tm’s near 70°C. The primers were added to a reaction mixture containing universal primers, TaqDNA polymerase, deoxynucleotide triphosphates, and buffer and RACE-PCR conducted as follows: (a) 5 cycles 94°C–30 s, 72°C–3 min; (b) 5 cycles 94°C–30 s, 70°C–30 s, 72°C–3 min; and (c) 25 cycles 94°C–30 s, 68°C–30 s, 72°C–3 min. PCR products were isolated by gel electrophoresis, cloned into PCR2.1/TOPO, amplified, and the isolated inserts sequenced. The HRPAP20 inserts were reamplified by 5'-RACE-PCR using gene-specific primers, resolved by agarose gel electrophoresis with the size verified by Southern blot analysis using the 32P-3'-289 bp cDNA, originally identified by differential display, as a probe. The nucleotide sequence was analyzed using MacVector 6.5 software (Oxford Molecular Group, Campbell, CA), which allowed identification of a single open reading frame (nucleotides 38–562) and the deduced amino acid sequence of a 20.2-kDa protein product.
The HRPAP20 cDNA was ligated into the HindIII (5') and EcoRI (3') multiple cloning site of the pcDNA 3.1 expression vector (Invitrogen, San Diego, CA) containing a cytomegalovirus promoter for constitutive expression corresponding to the 5'-end and an internal EcoRI site identified downstream of the open reading frame. After amplification in competent cells, the expression vector was isolated. The presence of the HRPAP20 insert was verified by HindIII and EcoRI digestion. The construct was then transfected into MCF-7 cells using lipofection (Promega, Madison, WI) and into Nb2-11 cells by electroporation. Control Nb2-11 and MCF-7 cells were transfected with an empty pcDNA 3.1 vector. Stable transfectants were selected using G418 (Calbiochem, San Diego, CA).
Generation of 
-HRPAP20 Antisera.
The HRPAP20 cDNA was subcloned into pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ). This construct was used for transformation of the competent Escherichia coli strain, BL21 DE3. For the initiation of HRPAP20 overexpression, the cultures were incubated for 3 h at 37°C with 0.5 mM isopropyl-1-thio-ß-D-galactopyranoside. The transformed cells were collected by centrifugation (6000 x g) and lysed by the freeze-thaw method in 1% Triton X in PBS, followed by sonication. The lysate obtained was centrifuged at 3500 x g and the supernatant filtered through a 0.22-µm filter and applied to a glutathione 4B resin column. The HRPAP20-GST fusion protein was eluted from the column and glutathione S-transferase removed using thrombin. The resulting purified HRPAP20 was concentrated to 1 mg/ml and used as an antigen for generation of polyclonal rabbit -HRPAP20 antisera ( Diagnostic International, San Antonio, TX). The antibody was additionally purified using the Affi-Gel Protein A MAPS II kit (Bio-Rad, Hercules, CA) and characterized by dot blotting with the purified antigen and cell lysates. Specificity for HRPAP20 was verified by immunoadsorption, immunoprecipitation with neutralized antibody, and comparison to preimmune serum. The working dilution for the antisera was determined to be 1:10,000 for immunoblotting and 2 µg for immunoprecipitation.
Northern Blot Analysis.
Total RNA was isolated from Nb2 lymphoma and MCF-7 cells using RNAzol-B (Tel-Test, Friendswood, TX) and quantitated spectrophotometrically. The RNA was denatured in formaldehyde and fractioned on 1% agarose gels, then transferred to GeneScreen Plus (DuPont, Wilmington, DE). Equal loading/lane was verified by densitometric analysis of ethidium bromide stained 18S and 28S rRNA, which was visualized and photographed under UV illumination. The 3'-289 bp HRPAP20 fragment was labeled with 32P-dCTP (New England Nuclear) using the random primer method of Feinberg and Vogelstein (17)
. Hybridization and wash procedures were conducted using the methods of Church and Gilbert (18)
.
Real-Time Quantitative PCR.
Total RNA was isolated from MCF-7 cells cultured in 1% insulin, transferrin, and selenium-positive DMEM using RNAzol-B. Oligonucleotide primers (HRPAP20: 5'-GTGCCT TCCTTG CCGGTAA-3', forward; and 5'-TCCTATTGGCAATCTGAACTCCTT-3', reverse) and probes (6FAM, reporter dye) specific for HRPAP20 and ß-actin were designed for use in TaqMan real-time PCR using Primer Express software (PE Applied Biosystems, Foster City, CA). A reaction mixture was prepared containing primers and probes at optimized final concentrations to which template cDNA was added in triplicate wells of optically clear 96-well reaction plates. An ABI PRISM 7000 Sequence Detection System was used to measure fluorescence during each PCR cycle [50°C for 2 min and 95°C for 10 min, followed by 15 s at 95°C (melting step) and 1 min at 60°C (anneal/extension) for 40 cycles]. Data were analyzed using a relative quantitation technique using sequence detection systems software. Each sample was normalized to levels of ß-actin mRNA within each sample. The mean level of expression from each sample was then compared with an internal reference.
Thymidine Incorporation.
Quiescent Nb2-11 cells were treated with ovine PRL (20 ng/ml) and harvested at 1, 2, 4, 6, 8, and 12 h for determination of HRPAP20 mRNA expression by Northern blot and densitometric analysis. Parallel cultures were pulse-labeled for 4 h with 0.5 µCi/well [3H]thymidine (SA, 86 Ci/mmol; Amersham Biosciences) and harvested at 4, 8, and 12 h.
To determine proliferation, quiescent Nb2-11 transfectants (3 x 104 cells/well) were cultured in PRL-free medium for 72 h; MCF-7 transfectants (6 x 103 cells/well) were plated in maintenance medium for 24 h and then cultured in insulin, transferrin, and selenium-supplemented DMEM for 96 h. The cultures were pulse labeled for 4 h with 0.25 µCi/well [3H]thymidine and harvested at the indicated times. At each time point, cells were harvested onto glass-fiber filters using an automated cell harvester (Cambridge Technology, Watertown, MA), precipitated with ice-cold 10% trichloroacetic acid and washed with ethanol. Radioactivity in trichloroacetic acid-insoluble material was determined by liquid scintillation spectroscopy.
Cell Proliferation Assay.
Nb2-11 cells, transfected with pcDNA 3.1 or the HRPAP20 cDNA were plated in 24-well plates in Fischer’s medium supplemented with 2-mercaptoethanol, antibiotics, and 10% nonmitogenic gelding serum, then exposed to increasing concentrations of PRL. Cells were counted electronically (Coulter, Hialeah, FL) after 48 h. Twenty-four-well plates were used to evaluate proliferation of MCF-7 transfectants exposed to increasing concentrations of PRL or estradiol in serum-free medium. Population density was determined after 96 h by electronic cell counting.
Immunoprecipitation/Immunoblotting.
Stationary Nb2-11 cells were incubated with PRL for 2, 4, 6, or 8 h. To evaluate regulation of HRPAP20 phosphorylation, protein kinase inhibitors H7 (6 µM), H8 (1.2 µM), or RO 31-8220 (10 nM) were added 0.5 h before the addition of PRL. The cells were rapidly cooled, centrifuged, then resuspended in a lysis buffer containing 10 mM Tris HCl (pH 7.4), 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, 5 µM phenylmethylsulfonyl fluoride, and 25 µg/ml each of aprotinin and leupeptin. The lysates were centrifuged for 30 min at 14,000 x g at 4°C. Total protein content was determined using the Bradford reagent (Bio-Rad). One mg of total protein was incubated with -HRPAP20 (2 µl) in the presence of protein G. After immunoprecipitation, proteins were resolved by SDS-PAGE on 12% gels and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Membranes were blocked overnight at 4°C in 5% nonfat dried milk in 0.05% Tween 20-PBS and then incubated with -HRPAP20 antibody (1: 10,000). For detection of phosphorylated HRPAP20, after immunoprecipitation, SDS-PAGE, and transfer, the membrane was blocked overnight at 4°C in 1% bovine serum albumin in PBS. Immunoblotting of the membranes was conducted using an -phosphoserine antibody (Calbiochem) to detect phosphorylated HRPAP20. Proteins were visualized by chemiluminescence detection using a secondary antibody coupled to horseradish peroxidase. Membranes were exposed to X-ray film for 5–15 min.
Analysis of Apoptosis.
A recognized characteristic of propidium iodide (PI)-stained apoptotic cells is the appearance of hypodiploid DNA (19)
. Control Nb2-11 and cells transfected with the empty vector or HRPAP20 were cultured in lactogen-free medium and harvested after 48 and 72 h. Aliquots of 1–2 x 106 cells were centrifuged at 200 x g and fixed by resuspension in cold 70% ethanol. The cells were washed, resuspended in cold PBS, then exposed to 1.0 mg/ml RNase. After 1 h at 37°C, the cells were washed and resuspended in a hypotonic PI solution (50 ng/ml). The stained cells were analyzed using an Elite flow cytometer (Coulter Electronics, Hialeah, FL) at 488 nm.
Presence of apoptotic cells was also assessed using the Vybrant Apoptosis Assay Kit 3 (Molecular Probes, Eugene, OR). Nb2-11 (1 x 106 cells/ml) transfectants were cultured in serum-free medium for 72 h then incubated with 5 µl of FITC-conjugated annexin V and PI for 15 min at room temperature. Fluorescence emission of the stained cells at 530 and >575 nm was analyzed using an Elite flow cytometer.
MCF-7 cells, transfected with the empty vector or HRPAP20 (2.5 x 104 cells/ml), were cultured in phenol red-free DMEM containing 1% insulin, transferrin, and selenium on microscopic culture slides (Falcon, Franklin Lakes, NJ). After 72 h of culture in the absence of serum, the cells were incubated with 300 µl/well of 2 µM calcein AM and ethidium homodimer-1 (Molecular Probes) for 30 min at room temperature. Viable cells were distinguished by the presence of green fluorescent calcein, because of intracellular esterase activity, which was retained. Nonviable cells took up ethidium homodimer-1, which upon binding to nucleic acids, produced a red fluorescence. Stained cells were photographed and counted in seven separate fields using a fluorescent microscope (Olympus CK40-RFL) and an attached videocamera (SpotInsight; Diagnostic Instruments, Inc., Sterling Heights, MI).
Statistical Analysis.
For Northern blotting procedures, equal loading/lane was verified by densitometric analysis of ethidium bromide staining of 18S and 28S rRNA. Where applicable, data are presented as the means ± SE. Differences among transfectants in experiments performed in Nb2-11 and MCF-7 cells were evaluated by ANOVA followed by the student Newman-Keuls posttest for multiple comparisons.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, the level of the 289-bp sequence expression was highest in malignant Nb2-SFJCD1 cells compared with quiescent Nb2-U17 or Nb2-11 cells. Its expression was reduced by treatment with the differentiating substance, NaBT, in the Nb2-SFJCD1 line. Using 5'-RACE-PCR, a 2117-bp rat HRPAP20 cDNA was cloned and its sequence introduced into GenBank. Computer-assisted sequence analysis of HRPAP20 revealed a single open reading frame that encoded a 20.2-kDa protein product (Fig. 2A)
. The sequence of the deduced rat HRPAP20 protein was found to be 81, 92, and 65% homologous to those of the human, mouse, and chick amino acid sequences, respectively (Fig. 2B)
.
|
|
helix (42–82% probability), random coil (17–40% probability), with an extended strand (0–13% probability).5
Motif-scan analysis of HRPAP20 with several databases (InterPro Scan prediction software) revealed the presence of a potential N-myristoylation sequence and several putative phosphorylation sites (Table 1)
. 6
, 7
Reinhardt’s method for cytoplasmic/nuclear discrimination suggested that HRPAP20 may reside within the nucleus (reliability 89%).8
|
, cells arrested in the G1 phase of cell cycle (0 h) expressed low levels of HRPAP20 compared with exponentially proliferating cultures (Log). Addition of a mitogenic concentration of PRL (20 ng/ml) rapidly increased accumulation of the HRPAP20 transcript. Increased levels were detected within 2–8 h (Fig. 3B)
after addition of the hormone. By 12 h, the HRPAP20 transcript returned toward unstimulated levels.
|
. As shown in Fig. 3A
, PRL-stimulated HRPAP20 expression was coincident with minimal [3H]thymidine incorporation from 2–8 h. However, the decline in HRPAP20 levels occurred simultaneously with increased [3H]thymidine incorporation as the cells entered S phase from 8–12 h. This kinetic pattern for PRL-stimulated Nb2-11 cell progression into S phase is consistent with our previous observations (20)
and suggests that hormone-induced expression of HRPAP20 is associated with progression through the G1 phase of cell cycle. Declining HRPAP20 occurs as cells begin synthesizing DNA.
Having demonstrated that HRPAP20 expression was stimulated by PRL in rat lymphoma cells, it was of interest to determine whether its expression was similarly affected in other PRL-responsive cell lines. Notably, recent studies have revitalized an interest in a possible contributory role for PRL in human breast cancer (13)
. The MCF-7 adenocarcinoma is a widely used paradigm for investigating hormone signaling and gene expression mechanisms in hormone-responsive breast cancer (21)
. However, because MCF-7 cells synthesize PRL endogenously (16)
, which may interfere with assessing hormonal actions in this cell line, we used MCF-7 cells that lacked endogenously synthesized PRL but retained hormone responsiveness. Stationary cells were cultured for 12 h in medium containing PRL (4 nM), which corresponds to the mid-G1 phase of the cell cycle in these cells under the conditions used (16)
to determine the effect of hormone on expression of HRPAP20 (Fig. 3D)
. Addition of PRL significantly (P < 0.05) increased HRPAP20 levels compared with control cells cultured in the absence of hormone (Fig. 3E)
. Thus, PRL-stimulated expression of HRPAP20 in human breast adenocarcinoma appears to be associated with cell cycle progression just as in the rat Nb2-11 cells.
Shown in Fig. 3F
is the characterization of the -HRPAP20 antisera used to evaluate HRPAP20 protein expression (Fig. 3D)
. Nb2-11 (left panel) and MCF-7 (right panel) cell lysates were resolved by SDS-PAGE and immunoblotted with -HRPAP20 (Fig. 3F
, Lanes 1 and 3). HRPAP20 appeared as a band that migrated slightly slower than the 20-kDa marker. This is consistent with the predicted co/posttranslational modification (myristoylation, phosphorylation) of the deduced 20.2-kDa sequence derived from the cloned HRPAP20 cDNA. The HRPAP20 band was absent or greatly reduced when preimmune serum (Fig. 3F
, Lane 2) or immunoadsorbed -HRPAP20 (Fig. 3F
, Lane 4) were used as primary reagents, demonstrating the specificity of the antisera. Notably, the reduction in the staining intensity of several bands ranging from 40 to 60 kDa suggests additional complexity in the expression and regulation of HRPAP20.
Differentiating Substances Reduce HRPAP20 mRNA Levels.
Previous studies indicated that NaBT arrested growth and transiently reversed the PRL independence in Nb2-SFJCD1 cells (2)
. Northern blot analysis was used to determine whether HRPAP20 expression was altered by exposure to NaBT or other diet-derived differentiating agents. Shown in Fig. 4A
is the effect of NaBT (2 mM, 72 h) on HRPAP20 mRNA expression in Nb2-SFJCD1 cells. Exposure of the cells to the differentiating agent markedly reduced HRPAP20 to nearly undetectable levels (time 0) compared with those observed in exponentially proliferating cultures (log; P < 0.0001). The addition of PRL, which stimulates mitogenesis under these conditions (2)
, stimulated rapid accumulation of the HRPAP20 transcript, again with maximal levels observed within 4–6 h before it declined toward controls by 8 h. Subsequently, it appeared to rise toward a second peak from 12 to 24 h in these cells.
|
; P < 0.005 and Fig. 4C
; P < 0.05, respectively). In each case, stimulation with PRL provoked rapid accumulation of its transcript with a temporal pattern nearly identical to that observed in hormone-stimulated Nb2-11 and NaBT-pretreated Nb2-SFJCD1 cells. To test the effect of dietary differentiating agents on HRPAP20 expressed in human breast cancer cells, MCF-7 cells were exposed to NaBT (2 mM) or RA (10 µM) for 48 or 72 h, respectively, before determination of HRPAP20 expression. Here, gene expression was determined by quantitative real-time PCR. As shown in Fig. 4E
, the differentiating agents significantly (P < 0.05) reduced expression of HRPAP20 compared with untreated cells in a manner similar to that observed in the malignant rat lymphoma line. Taken together, these results indicate that HRPAP20 expression is a characteristic of exponentially proliferating rat Nb2-SFJCD1 cells and human MCF-7 cells. Exposure of these cell lines to dietary differentiating substances, which suppress growth, reduced its expression. In Nb2-SFJCD1 cells exposed to these compounds, mitogenic stimulation with PRL increased HRPAP20 expression.
HRPAP20 Protein Levels and Its Phosphorylation.
Because HRPAP20 mRNA expression was regulated by hormones and differentiating agents, we assessed whether its protein product was similarly modulated in Nb2-11 cells. Immunoblot analysis revealed that exposure of quiescent Nb2-11 cells to PRL significantly increased HRPAP20 protein levels between 2 and 6 h (Fig. 5A)
. By 8 h, its level returned toward that observed in untreated controls. Densitometric analysis (Fig. 5B)
confirmed that the pattern of PRL-stimulated HRPAP20 protein expression was similar to that observed for the effect of the hormone on accumulation of its transcript.
|
. Quiescent Nb2-11 cells were used to investigate whether HRPAP20 was a substrate for phosphorylation. Lysates of PRL-treated cells were immunoprecipitated with -HRPAP20, then evaluated by immunoblotting using a highly specific -phosphoserine monoclonal antibody that recognized serine residues followed by proline or lysine. As shown in Fig. 6A
, HRPAP20 appeared to be phosphorylated on serine residues during a PRL-mediated growth response. Increased serine phosphorylation was detected within 1.5–6 h after the addition of PRL, then declined by 8 h (Fig. 6B)
. Inhibition of phosphorylation at serine residues was evaluated using the pharmacological inhibitors H7 (PKA, PKC, PKG), H8 (PKA), and RO 31-8220 (PKC) before PRL treatment (Fig. 6C)
. Densitometric analysis revealed that RO 31-8220 significantly reduced the phosphorylation of HRPAP20 (P < 0.05, Fig. 6D
).
|
or untransfected control Nb2-11 cells (data not shown). In a companion experiment, MCF-7 transfectants were cultured under serum-free conditions for 96 h. Here, just as with Nb2-11 cells, expression of HRPAP20 significantly (P < 0.006) increased the population density of MCF-7 cells by >50% (Fig. 7C)
. The effect of HRPAP20 expression to increase proliferation was additionally evaluated by measuring [3H]thymidine incorporation. As demonstrated in Fig. 7, B and D
, HRPAP20 expression significantly increased [3H]thymidine incorporation in Nb2-11 and MCF-7 cells. Because only viable cells synthesize DNA, into which [3H]thymidine is incorporated, we conclude that HRPAP20 increases tumor cell proliferation in the absence of hormone/growth factor stimulation. The effect of PRL and PRL or estradiol on Nb2-11 and MCF-7 transfectant proliferation, respectively, was assessed in other experiments. Although each of the transfected cell lines grew more rapidly in the absence of hormonal stimulation, no consistent differences were observed with PRL or estradiol treatment (data not shown). These results indicate that increased HRPAP20 expression facilitates cell proliferation and suggests that this gene may encode an important regulator of the growth response.
|
, the percentage of hypodiploid cells in untransfected control Nb2-11 and empty vector transfectants were 50% after 48 h (Fig. 8A)
and >65% by 72 h (Fig. 8B)
. However, constitutive expression of HRPAP20 significantly reduced the percentage of cells undergoing apoptosis at each time point, suggesting that HRPAP20 expression renders cells less susceptible to cell death induced by prolonged lactogen deprivation (Fig. 8, A and B)
. The extent of apoptosis was additionally measured using FITC-conjugated annexin V labeling of PI-stained cells. Flow cytometric analysis showed that Nb2-11 cells transfected with the empty vector exhibited a significantly (P < 0.01) greater percentage of apoptotic cells compared with levels observed in HRPAP20transfectants 72 h after serum starvation (Fig. 8, C–E)
. The effect of HRPAP20 on MCF-7 cell survival was also investigated. As shown in Fig. 8F
, MCF-7 cells constitutively expressing HRPAP20 exhibited a significantly greater number of viable cells and fewer dead cells compared with empty vector transfectants subsequent to culturing for 72 h in serum-free medium. Taken together, these results suggest that HRPAP20 expression may afford a survival advantage to serum-starved tumor cells.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Differential display is a fingerprinting technology that facilitates identification of mRNAs that exhibit altered expression (22)
. A significant number of differentially expressed genes have been identified using this approach since it was initially developed (23)
. Using this strategy, we identified HRPAP20 as a novel gene that was differentially expressed in exponentially growing rat Nb2-11 lymphoma cells and its highly malignant subline, Nb2-SFJCD1. Cloning and subsequent sequence analysis of HRPAP20 revealed a single open reading frame that encoded a putative 20.2-kDa protein. The significant amino acid sequence homology of rat HRPAP20 with those deduced for human, mouse, and chick demonstrates its high conservation among species (Fig. 2B)
. Furthermore, computer-assisted structural analysis suggested that HRPAP20 is likely a globular N-myristoylated protein potentially located within the nucleus, which may be regulated by phosphorylation.
The rat Nb2-11 lymphoma is a hormone-dependent cell line that exhibits a critical growth and survival requirement for PRL. Cell synchronization in G1 by lactogen withdrawal in Nb2-11 cells profoundly reduced HRPAP20 expression. Mitogenic stimulation of quiescent Nb2-11 cells increased accumulation of its mRNA. Pulse-labeling experiments with [3H]thymidine indicated that PRL stimulated accumulation of HRPAP20 mRNA as the cells traversed through the G1 phase of cell cycle, followed by its return toward basal levels upon entry into S phase. Additional evidence for a role for HRPAP20 during a growth response was obtained in experiments in which its expression was assessed in MCF-7 cells. Because MCF-7 cells express endogenous PRL, the MCF-7/GAS3TK cell line was developed in which PRL expression was prevented; however, the cells retained PRL responsiveness (16) . Treatment of MCF-7/GAS3TK cells with PRL, which accelerates growth (24) , significantly increased HRPAP20 levels by 12 h. Protein levels of HRPAP20 were low in untreated MCF-7/GAS3TK cells cultured in the absence of serum similar to the effect of PRL withdrawal in Nb2-11 cells. In each cell line, HRPAP20 expression was decreased in the absence of PRL; addition of the hormone increased its accumulation.
Expression of HRPAP20 was also regulated by exposure to substances that induce differentiation. NaBT, RA, and vitamin D3 are examples of diet-derived substances that induce differentiation and, as a result, alter specific gene expression in tumors in vivo and in cell culture. Butyrate, a short chain fatty acid, is physiologically produced by bacterial fermentation of dietary fiber in the colon. It was shown that NaBT inhibited proliferation by favoring cell cycle arrest and promoting differentiation in normal and transformed cells (25) . In addition, NaBT induced apoptosis in certain tumor cell lines (26) . On a molecular level, it alters expression of genes associated with proliferation and differentiation, primarily by inhibiting histone deacetylase (27) .
Another diet-derived differentiating substance that has demonstrated efficacy in numerous cancers is RA. Breitman et al. (28)
were the first to report that RA induced differentiation in hematopoietic cells. Moreover, promising clinical results have been obtained in treatment of acute promyelocytic leukemia with RA, a disease characterized by chromosomal translocations involving the RA receptor and promyelocytic leukemia genes (29)
. In breast cancer, preclinical studies showed that retinoids inhibited chemically induced carcinogenesis in rat models and suppressed growth of certain human breast carcinoma cell lines (30)
. In the MCF-7 line, RA inhibited growth and increased cell adhesion to fibronectin (31)
. This effect was observed in estrogen receptor-positive breast tumor lines but not in those lacking the receptor. RA mediates its cellular effects via RA receptors (RAR and RXR), members of the nuclear hormone receptor family (32
, 33)
.
As with RA, vitamin D3 also induces differentiation in tumor cells via an interaction with a nuclear hormone receptor, although a direct effect on phosphatidylinositol-3-kinase also has been reported (34) . The vitamin D3 receptor acts as a heterodimer with the RXR, suggesting potential interactions between retinoids and vitamin D analogues. Thus, NaBT, RA, and vitamin D3 each exhibit potent growth inhibitory effects and render hematopoietic and hormone-responsive breast cancer cell lines sensitive to induction of apoptosis.
The effect of these dietary-differentiating substances on HRPAP20 expression was evaluated in Nb2-SFJCD1 and MCF-7 cells. Karyotypic analysis of Nb2-SFJCD1 cells showed that these cells arose from the parental line by clonal evolution and represent a more advanced stage of malignant progression compared with the original Nb2 lymphoma (35) . Treatment of Nb2-SFJCD1 cells with NaBT, RA, or vitamin D3 decreased expression of HRPAP20 and transiently reversed PRL independence of these cells. Addition of PRL rapidly increased its mRNA levels, demonstrating hormone responsiveness under these conditions. These effects of the differentiating agents on HRPAP20 expression are strikingly similar to our previous observations of the effects of NaBT on c-myc and pim-1 expression in Nb2-SFJCD1 cells (36) . In human breast MCF-7 cells, NaBT and RA also reduced the level of the HRPAP20 transcript. The observation that each of these substances suppressed HRPAP20 expression in a rat Nb2 lymphoma and in human breast carcinoma cells suggests that HRPAP20 may be central to growth regulation, differentiation, or apoptosis sensitivity in hormone-responsive cancers.
Having observed hormonal regulation of HRPAP20 mRNA expression, it was important to determine whether its protein was similarly regulated. Immunoblot analysis verified that the HRPAP20 protein was regulated by PRL in a manner similar to that of its mRNA in Nb2 and MCF-7 cells. In each cell line, the PRL-stimulated protein expression occurred during G1 cell cycle progression, suggesting that HRPAP20 may be critical for cell growth. On the basis of computer-assisted analysis of the deduced HRPAP20 amino acid sequence, we conducted experiments to determine whether HRPAP20 was phosphorylated. Because the predicted HRPAP20 sequence contains several potential serine phosphorylation sites, immunoblot analysis using phosphoserine-specific antibodies was used to determine whether it is a substrate for phosphorylation. The results indicated that HRPAP20 was phosphorylated on serine residues in a PRL-dependent manner, indicating that it is most likely a substrate for kinase-catalyzed phosphorylation. However, it is notable that the kinetic pattern for PRL-stimulated HRPAP20 phosphorylation (Fig. 6)
was essentially identical to that observed for its protein expression (Fig. 5)
. Therefore, it is possible that the observed increase in HRPAP20 serine phosphorylation may reflect PRL-mediated accumulation of the constitutively phosphorylated protein. Several pharmacological inhibitors of common protein kinases were used to evaluate whether their antagonism altered phosphorylation of HRPAP20. At the concentrations used, only RO 31-8220 significantly reduced phosphorylation of HRPAP20 protein in Nb2-11 cells, an observation consistent with the prediction that HRPAP20 may contain several PKC-phosphorylatable sites. This observation suggests the possibility that HRPAP20 functional activity may be regulated by PKC. It is important to note that several previous studies have linked PRL signaling to activation of PKC in Nb2 cells and in the mammary gland (37, 38, 39)
. Thus, it is possible that at least one substrate for PRL-stimulated PKC is HRPAP20, which may serve as an intermediate in the regulation of downstream effects.
Expression of HRPAP20 and its phosphorylation were associated with a growth response; therefore, we hypothesized that its expression may regulate proliferation. Stable expression of HRPAP20 in Nb2-11 and MCF-7 cells significantly increased proliferation in the absence of mitogenic stimulation. Thus, HRPAP20 appears to be an important component of a growth response. It will be important to address the molecular mechanism(s) by which HRPAP20 modulates proliferation in future studies.
Constitutively elevated HRPAP20 increased the population density of cells cultured in the absence of serum for 72–96 h. Because prolonged growth factor deprivation stimulates cell death in responsive cells, it was important to determine whether its augmented expression affected apoptosis, a key process frequently dysregulated in tumors. Cells undergoing apoptosis display morphological and physical property changes (cell shrinkage, condensation of chromatin, and hydrolysis of DNA) that are measurable by flow cytometry (40) . The appearance of subdiploid cells is a quantifiable and specific marker of apoptosis (19) . Using this approach, we observed that HRPAP20 expression suppressed apoptosis induced by growth factor (PRL) deprivation in Nb2-11 cells, a potent stimulus of active cell death (41) . A role for an apoptosis-attenuating action of HRPAP20 was additionally evaluated by assessing loss of membrane asymmetry with respect to phosphatidylserine (PS) membrane localization, an additional characteristic of cells undergoing apoptosis (42) . In viable cells, phosphatidylserine is located on the cytoplasmic surface of the plasma membrane; however, in apoptotic cells, phosphatidylserine translocates to the outer membrane leaflet, thus exposing it to the external cellular environment (43) . This process can be measured using the human anticoagulant, annexin V, which binds with high affinity to phosphatidylserine, labeled with a fluorophore (FITC). Results obtained using this approach confirmed our previous observation that HRPAP20 increased survival in PRL-starved Nb2-11/HRPAP20 transfectants.
The effect of HRPAP20 on MCF-7 survival was also evaluated. The previous observations that endonucleolytic DNA fragmentation was not required for inducing chromatin condensation associated with apoptosis in MCF-7 cells (44) , taken together with the lack of caspase-3 expression due to a 47-bp deletion in exon 3 of CASP3 (45) in this cell line, led us to evaluate whether elevated HRPAP20 affected cell viability in serum-starved MCF-7 cells. Using a two-color fluorescence assay, viable and nonviable cells were distinguished based on their physical and biochemical properties. The results obtained from these experiments showed that constitutive HRPAP20 expression in MCF-7 cells significantly augmented survival in the absence of serum for 96 h, similar to its effects in Nb2-11 cultures. Taken together, these observations suggest that elevated levels of HRPAP20 are protective against cell death mechanisms. Therefore, the observations of increased cell density in HRPAP20 expressing Nb2-11 and MCF-7 cultures subsequent to prolonged serum starvation are most likely the result of HRPAP20-mediated augmentation of proliferation and suppression of cell death.
An important unanswered question that remains is how does HRPAP20 increase tumor cell growth or survival? Future studies will focus upon intracellular mechanisms of HRPAP20 regulation to additionally define the consequences of its expression. We suggest that elucidation of HRPAP20 mechanisms and actions may prove predictive of the clinical course of hormone-responsive or other tumor cells with respect to proliferative capacity or drug resistance. Moreover, it is possible that HRPAP20 may also provide a new therapeutic target for treatment of breast cancer or other malignancies.
|
FOOTNOTES |
|---|
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.
Notes: The present location for H. Pan is Arena Pharmaceuticals, Inc., San Diego, CA. The present location for M. Zhang is Department of Molecular Biology and Biochemistry, University of California, Irvine, CA.
Requests for reprints: Arthur R. Buckley, University of Cincinnati, College of Pharmacy, 3223 Eden Avenue, Cincinnati, OH 45267. Phone: (513) 558-2575; Fax: (513) 558-0978; E-mail: Arthur.Buckley{at}uc.edu
4 Internet address: http://occawonline.pearsoned.com/bookbind/bc_mccampbell_genomics. 
5 Internet address: http://cu.bic.bioc.columbia.edu.
6 Internet address: http://www.ebi.ac.uk.
7 Internet address: http://hits.isb_sib.ch.
8 Internet address: http://psort.nibb.ac.jp.
Received 1/ 7/03. Revised 10/29/03. Accepted 11/10/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
gene is rearranged in retinoic acid-sensitive promyelocytic leukemias. Leukemia (Baltimore), 4: 802-807, 1990.
,25-dihydroxyvitamin D3-induced myeloid cell differentiation is regulated by a vitamin D receptor-phosphatidylinositol 3-kinase signaling complex. J. Exp. Med., 190: 1593-1594, 1999.
This article has been cited by other articles:
|
|
 |
|
  S Tozlu-Kara, V Roux, C Andrieu, J Vendrell, S Vacher, V Lazar, F Spyratos, M Tubiana-Hulin, P Cohen, P Dessen, et al. Oligonucleotide microarray analysis of estrogen receptor {alpha}-positive postmenopausal breast carcinomas: identification of HRPAP20 and TIMELESS as outstanding candidate markers to predict the response to tamoxifen J. Mol. Endocrinol., October 1, 2007; 39(4): 305 - 318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Dusso, A. J. Brown, and E. Slatopolsky Vitamin D Am J Physiol Renal Physiol, July 1, 2005; 289(1): F8 - F28. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
