Leukocyte common-antigen-related tyrosine phosphatase receptor: altered expression of mRNA and protein in the New England Deaconess Hospital rat line exhibiting spontaneous pheochromocytoma

Tao Yang, John A. Martignetti1, Stephen M. Massa and Frank M. Longo2

Department of Neurology, University of California San Francisco/VA Medical Center, 4150 Clement Street, San Francisco, CA 94121 and
1 Department of Human Genetics and Pediatrics, Mount Sinai Medical Center, New York, NY 10029, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Regulation of cell proliferation by protein tyrosine phosphatases (PTPs) suggests that PTPs are important tumor suppressor genes. The gene encoding the leukocyte common-antigen-related (LAR) PTP receptor maps to chromosome 1p32–33, a region in which loss of heterozygosity is associated with human pheochromocytoma and other neuroectodermal tumors. The rat pheochromocytoma PC12 cell line was originally derived from the transplantable P259 tumor originating from the New England Deaconess Hospital (NEDH) line of Wistar inbred rats. Compared with their Wistar counterparts, 1–2-year-old NEDH rats exhibit a high incidence of spontaneous pheochromocytomas. This study investigates whether levels of LAR transcripts and protein are altered in NEDH adrenal tissue prior to tumor onset. In addition, alternative splicing of an LAR extracellular domain [LAR alternatively spliced element-c (LASE-c)], regulating LAR interaction with extracellular matrix components, was examined. These changes in LAR expression and alternative splicing were hypothesized to be more pronounced in tumor tissue and PC12 cells. Northern blot analysis demonstrated the presence of the ~5 kb LAR transcript in all cell lines examined, except PC12. In adrenal medulla tissue harvested from 2–3-month-old rats, LAR ~8 and ~5 kb transcript expression was decreased in NEDH compared with Wistar samples. RT–PCR demonstrated increased splicing of the LASE-c 27 bp alternatively spliced insert in the LAR extracellular domain in NEDH adrenal medulla tissue. Even greater LASE-c splicing was detected in adrenal medulla tumor tissue derived from 12-month-old NEDH rats and in PC12 cells. Western blot analysis demonstrated decreased levels of LAR protein and increased levels of LASE-c containing LAR protein isoforms in NEDH adrenal medulla tissue. These studies demonstrate that patterns of altered LAR expression present in PC12 cells and in pheochromocytoma tumor tissue are also present in adrenal tissue predisposed to a high incidence of spontaneous pheochromocytoma.

Abbreviations: 3'-UTR, 3'-untranslated region; LAR, leukocyte common-antigen-related; LASE-c, LAR alternatively spliced element-c; PTKs, protein tyrosine kinases; PTPs, protein tyrosine phosphatases.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The discovery that many oncogenes consist of either protein tyrosine kinases (PTKs), their ligands or their targets, and the observation that protein tyrosine phosphatases (PTPs) can counterbalance actions of PTKs, led to the hypothesis that certain PTP genes might constitute tumor suppressor genes (1). The finding that expression of certain PTPs was upregulated in various tumors provided initial support for this hypothesis and identified specific PTPs as candidates that might influence transformation and/or tumor growth (26). Introduction of PTP1B into NIH 3T3 cells suppressed oncogenic transformation by the neu gene (7). Ectopic expression of PTP1B also inhibited tumor formation by p210 bcr-abl-transformed fibroblasts (8). Introduction of the leukocyte common-antigen-related (LAR) PTP into a human breast carcinoma cell line that overexpresses the p185neu PTK decreased proliferation rate in vitro and suppressed tumor growth following inoculation in vivo (9). Similarly, introduction of the Dep-1 PTP into human breast cancer cell lines inhibited cell growth 5–10-fold (10). Expression of p164PTP-TD14 in NIH 3T3 cells inhibited Ha-ras-mediated transformation (11). The hypothesis that PTPs can act as tumor suppressors was further supported by the discovery that inactivation mutations in the PTEN PTP gene are associated with human cancers including human breast, prostate and thyroid cancer (12,13).

Identification of PTPs demonstrating altered expression in tissues pre-disposed to tumor formation in early or pre-tumor stages will provide additional critical data for identifying candidate PTPs influencing tumor formation. Several features of the LAR PTP make it of particular interest in evaluating PTP mechanisms in neuroectodermal tumor formation. LAR is a prototype member of the superfamily of receptor PTPs containing Ig- and fibronectin type III-like cell adhesion motifs in their extracellular region (14). Alternative splicing of LAR transcripts is developmentally regulated and occurs preferentially in the nervous system (1517). Alternative splicing of a 27 bp insert [LAR alternatively spliced element-c (LASE-c)] in the extracellular domain of LAR regulates LAR interaction with laminin–nidogen complexes (18). Transgenic mice and Drosophila mutants deficient in LAR expression demonstrate aberrant neural networks consistent with the hypothesis that LAR regulates specific cell adhesion interactions (19,20). In the PC12 pheochromocytoma cell line, inhibition of LAR expression by stable transfection with LAR antisense transgenes causes increased proliferation rates (21). The human LAR gene is located at chromosome 1p32–33. Genetic alterations in the distal region of the short arm of chromosome 1 have been associated with multiple neuroectodermal tumors (22), including pheochromocytoma, neuroblastoma, astrocytoma and malignant melanoma (2328). It is of particular interest to note that ~10% of human pheochromocytomas are hereditary and that aberrations near 1p32–33 are frequently associated with these tumors.

To address the possibility that LAR might constitute a PTP influencing oncogenic transformation or early tumor cell proliferation, the occurrence of aberrant splicing or decreased expression of LAR in tissue predisposed to tumor formation was examined. The New England Deaconess Hospital (NEDH) strain of Wistar rat is one of a handful of inbred Wistar strains that demonstrate a markedly increased incidence of spontaneous pheochromocytoma with reported rates of 59% in male rats 700–900 days old (29,30). In these strains, the increase in tumor rate occurs primarily after 1 year of age (3032). In the more commonly available Wistar strains, pheochromocytoma rates are typically 5–10% with a range of 1–22% observed over the first 2 years of life (33). The development of spontaneous pheochromocytomas in NEDH rats has led to their use as an animal model for spontaneous hypertension (30). The finding that exposure of NEDH rats to radiation further increased the incidence of these tumors led to the establishment of the P259 transplantable pheochromocytoma (29) and the P259-derived PC12 pheochromocytoma cell line (34).

Preliminary studies in our laboratory indicated that one LAR mRNA isoform (~5 kb) was expressed in a large number of neuroectodermal-derived cell lines but was undetectable in the PC12 cell line. This observation led to the hypothesis that LAR expression and/or alternative splicing might also be altered in adrenal medullary tissue predisposed to pheochromocytoma formation, and that these alterations would be even more pronounced in tumor tissue. This hypothesis was addressed by comparing LAR expression and alternative splicing in Wistar and Wistar-NEDH adrenal medullary tissue harvested well before the age of tumor onset and in adrenal medullary tissue harvested from older rats showing morphological features of tumor. It was also hypothesized that changes in alternative splicing in pre-tumor tissue might resemble or predict changes found in tumor tissue and in PC12 cells. Detection of altered LAR expression and/or transcript splicing in tissue predisposed to tumor formation would encourage further studies examining the role of LAR in neuroectodermal tumor formation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Wistar and NEDH rats
Wistar and NEDH rats were obtained from Simonsen Laboratories (Gilroy, CA). Simonson Wistar rats were originally derived from Wistar rats obtained from the University of Notre Dame and have been bred as a closed colony since 1957. NEDH rats were originally derived through inbreeding of Wistar rats by S.Warren at the NEDH (29,30) and introduced to the Simonsen Laboratories in 1987 via B.Hoffman (Veterans Administration Medical Center, Palo Alto, CA) (35). Three breeding pairs of NEDH rats were crossed to produce F1 progeny. To assess LAR expression during pre-tumor stages, Wistar and NEDH F1 rats at the ages of 2–3 and 10–12 months were used for harvesting tissues. A group of eight NEDH rats were maintained for 12–24 months in order to confirm the appearance of spontaneous pheochromocytomas in the line of NEDH rats obtained for the present study.

Hematoxylin and eosin staining
Frozen sections of adrenal tissue (5 µ thick) were fixed in methanol for 10 min, rinsed in water twice, stained with hematoxylin (Sigma, St Louis, MO) for 1 min, rinsed in Scott's water (Sigma) for 2 min and washed with water for 2 min. Sections were then treated in 80% ethanol for 2 min, stained with eosin (Sigma) for 2 min, rinsed with 95% ethanol three times, treated with 100% ethanol for 3 min and xylene for 5 min followed by application of coverslips.

Cell cultures
PC12 cells (36) (obtained from Dr William Mobley, UCSF) were grown in Dulbecco's modified Eagle's Medium (DMEM; with 3.7 g/l NaHCO3, 4.5 g/l glucose, 0.584 g/l glutamine) containing 5% fetal bovine serum, 10% horse serum and penicillin–streptomycin in untreated tissue culture flasks. Primary rat astrocyte cultures were prepared from postnatal day two rat cortex as described previously (37). Cortical cultures were enriched for astrocytes by differential attachment. Oligodendrocyte O2A precursor cells were cultured as described previously (38). Astrocytes and O2A cell pellets were provided by Drs E.Magal and J.C.Louis (37,38). COS cells and Swiss 3T3 fibroblasts (American Type Culture Collection, Rockville, MD) were grown in DMEM with 10% fetal bovine serum. P19 cells were grown in MEM {alpha} with nucleosides (Gibco BRL, Grand Island, NY) and 10% fetal bovine serum. All cells were harvested when they reached 90–100% confluence and stored at –70°C.

Isolation of RNA and northern blot analysis
Poly(A) RNA was directly isolated from tissues and cell pellets by incubation of cell pellets or tissue samples in lysis buffer with oligo(dT) cellulose using a standard protocol and regents [FastTrack poly(A) RNA isolation kit; Invitrogen, San Diego, CA]. To remove any contaminating DNA, poly(A) RNA was incubated with DNase (2 U in a total volume of 400 µl; Promega Biotech, Madison, WI) at 37°C for 30 min. Northern blot hybridization was carried out using standard protocols (39). Poly(A) RNA (4 µg per lane) was electrophoresed through 1.0% formaldehyde/agarose gels and transferred to nylon membranes (Hybond-N+, Amersham, Arlington Heights, IL). LAR riboprobe corresponding to the constitutive LAR 3'-untranslated region (3'-UTR) was prepared from rat pLM65-10 cDNA clone as described previously (40). This probe primarily identifies the ~8 kb full-length LAR transcript. LAR riboprobe corresponding to the alternatively spliced LAR 3'-UTR was synthesized using the pLARAccI cDNA clone as described previously (15). This probe corresponds to an ~900 bp stretch located downstream from the CAG repeat region located in the alternative 3'-UTR and identifies an ~5 kb LAR transcript. Cyclophilin riboprobe was synthesized with SP6 RNA polymerase using rat cyclophilin clone plB15 linearized with PstI (41).

Hybridizations were performed overnight at 50°C using riboprobe in 50% formamide, 5x Denhardt's, 5x SSPE, 1% SDS and 100 µg/ml boiled salmon sperm DNA. Washes were carried out under high stringency conditions: 2x SSC/0.1% SDS three times for 5 min each at room temperature, treated with RNaseA (1 µg/ml; Promega Biotech) in 2x SSC for 5 min at room temperature, washed in 0.1% SDS/0.1x SSC at 50°C for 30 min, and then in 0.1% SDS/0.1x SSC at 65°C for 30 min. Blots were exposed to X-ray film for 30–60 min for cyclophilin detection and 1–4 days for LAR detection. Northern blot autoradiographs were densitometrically scanned using the Bethesda Research Laboratory imaging system.

RT–PCR
Total RNA was isolated from adrenal tissue using STAT-60 solution (Tel-Test `B' Inc., Friendswood, TX). To remove any contaminating DNA, RNA was incubated with DNase (2 U in a total volume of 400 µl; Promega Biotech) at 37°C for 30 min. Quantitative (RT–PCR) analysis and the primers used for measuring the relative proportions of LAR transcripts containing the 27 bp LASE-c insert have been described previously (15). PCR primers flanking the LASE-c inserts were used for RT–PCR reactions. First-stand cDNA synthesis with total RNA and random hexamers and subsequent PCR were conducted using reagents and protocols included in the GeneAmp RNA PCR kit (Perkin Elmer-Cetus, Norwalk, CT). PCR was performed for 35 cycles as follows: denaturing, 95°C for 1 min, annealing at 67°C for 1 min, extension at 72°C for 1 min and final extension step at 72°C for 10 min. All RT–PCR and PCR reaction sets included the use of water in place of template in one or more reactions to serve as a negative control.

RT–PCR products were electrophoresed through 6% polyacrylamide gels, stained in ethidium bromide and photographed. Photographic negatives were scanned by densitometry and the ratio of band signal associated with PCR product with and without the LASE-c insert was calculated. Since amplification efficiencies of PCR products using the same primers and amplifying products differing in size by less than several hundred base pairs remain similar in both the exponential and plateau phase of the amplification reaction, the change in the ratio of the products can be used to measure changes in the relative proportions of the starting transcripts (42). The identification of PCR products with and without the LASE-c insert was confirmed by subcloning and sequencing (15).

Western blot analysis
Affinity-purified antibody raised against a peptide corresponding to the LASE-c insert has been characterized previously (15,16). Monoclonal antibody directed against the LAR N-terminus was purchased from Transduction Laboratories Inc. (Lexington, KY). Antibodies directed against the LAR N-terminus and LASE-c identify the LAR ~150 kDa extracellular subunit (16). Monoclonal antibody directed against actin was purchased from Sigma.

Protein extracts were prepared from tissue samples and cell pellets by lysis in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 µg/ml antipain, 1 µg/ml pepstatin, 2 mM phenylmethylsulfonyl fluoride) for 30 min on ice, sonicated for 10 s, and centrifuged at 14 000 g for 30 min. Supernatant was collected and concentrated using a Centricon-30 filter (Amicon, Beverly, MA). Protein extract (100 µg) was electrophoresed through a 7.5% polyacrylamide gel and transferred to PVDF membranes (Amersham). Blots were incubated overnight with primary antibody followed by secondary antibody-based detection using the ECL Detection System (Amersham).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Confirmation of spontaneous pheochromocytomas in NEDH rats
Wistar and NEDH rats obtained from Simonson Laboratories were killed for autopsy at 12–24 months of age and adrenal glands harvested. Histological analysis of adrenal tissue isolated from two NEDH rats (#274 and #331) with enlarged adrenal glands at 12 months of age was conducted. In tissue from both rats multiple clusters of basophilic small cells were found in the medulla (Figure 1Go). This pattern of cell aggregation is typical of early stage pheochromocytoma in the rat (30). Adrenal tumors were not detected in Wistar rats.



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Fig. 1. Histological analysis of NEDH adrenal tissue. Hematoxylin and eosin staining of adrenal frozen sections harvested from NEDH rat #274 at 12 months of age demonstrated clusters of basophilic small cells in the medulla (arrows). The adrenal cortical region is evident in the more darkly staining area of the upper right corner. Magnification x400.

 
LAR transcript expression in PC12 and other cultured cells
Northern blot analysis was used to compare levels of LAR ~8 and ~5 kb transcripts in PC12 and other cells. Similar levels of LAR ~8 kb transcripts were readily detected in PC12 cells and each of the other five cell types examined (Figure 2AGo). The expression of LAR in the PC12 and P19 neural cell lines, astrocytes, the O2A oligodendrocyte cell line, COS cells and 3T3 fibroblasts was consistent with its expression across a wide range of tissue types in vivo (40). In contrast to the ~8 kb full-length, ~5 kb LAR isoforms were not detected in PC12 cells while clearly apparent in the other five cell types (Figure 2BGo). The lack of ~5 kb signal in PC12 cell samples was found in three separate northern analyses. This finding suggested the hypothesis that alterations in LAR expression might be present in NEDH adrenal medullary tissue.



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Fig. 2. Northern blot analysis of LAR ~8 and ~5 kb transcripts in cultured cells. Poly(A) RNA (4 µg loaded per lane) was isolated from the following cell lines: PC12 cells, P19 neuroectodermal cells, NIH 3T3 fibroblasts, COS cells and O2A oligodentrocytes. Primary cultures of astrocytes (Astro) were prepared from E16 rat cortex. (A) Blots were probed with riboprobe corresponding to the LAR constitutive 3'-UTR. All six cell types expressed the ~8 kb LAR transcript (upper panel). (B) Blots were probed with riboprobe corresponding to the LAR alternative 3'-UTR. All cell types except PC12 cells expressed the ~5 kb LAR isoform (upper panel). Blots were stripped and reprobed with cyclophilin (cyc) riboprobe (lower panels). RNA size markers (kb) are shown on the left of each blot.

 
LAR transcript expression is decreased in NEDH adrenal tissue
Poly(A) RNA was isolated from adrenal medullar and cortical tissue harvested from Wistar and NEDH rats. For each strain, RNA preparations were derived from pooled tissue obtained from a total of six (three male and three female) 2–3-month-old rats. Northern blot analysis (Figure 3A and BGo) using the constitutive LAR 3'-UTR riboprobe demonstrated that relative to cyclophilin signal, LAR ~8 kb transcript levels were decreased in NEDH adrenal medulla to 28 ± 16% of Wistar levels, and in NEDH adrenal cortex to 56 ± 15% of Wistar levels. Taken together, ~8 kb transcript levels in NEDH medulla and cortex were 42% of that found in Wistar (P < 0.01, n = 6 northern blots, Mann–Whitney test). Northern blot analysis using the alternative 3'-UTR riboprobe (Figure 4A and BGo) revealed a significant decrease in levels of the LAR ~5 kb transcript in NEDH adrenal medulla to 20 ± 2% of Wistar levels. In NEDH adrenal cortex, levels were decreased to 59 ± 5% of Wistar levels although this decrease did not reach significance.



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Fig. 3. Northern blot analysis of LAR ~8 kb transcripts in Wistar (WIST) and NEDH rat adrenal tissue. (A) Poly(A) RNA (4 µg loaded per lane) was isolated from Wistar and NEDH adrenal cortical and medullary tissues. Pairs of adrenal tissues were combined from six Wistar and six NEDH rats and one poly(A) RNA preparation prepared from each pool. Blots were probed with riboprobe corresponding to the LAR constitutive 3'-UTR (upper panel). Blots were stripped and reprobed with cyclophilin (cyc) riboprobe (lower panel). RNA size markers (kb) are shown on the left of the blot. (B) Autoradiographs from three independent northern blot studies were analyzed by scanning densitometry and the ratio of signal from the ~8 kb band over the signal from the cyclophilin band was calculated. Mean ratios ± SE (n = 3 northern blots) are shown. Values obtained from each blot were normalized against the average ratio obtained for Wistar adrenal cortex.

 


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Fig. 4. Northern blot analysis of LAR ~5 kb transcripts in Wistar (WIST) and NEDH rat adrenal tissue. (A) Poly(A) RNA (4 µg loaded per lane) was isolated from Wistar and NEDH adrenal cortical and medullar tissues. Pairs of adrenal cortexes and medullas were combined from six Wistar and six NEDH rats and one poly(A) RNA preparation prepared from each pool. Blots were probed with riboprobe corresponding to the LAR alternatively spliced 3'-UTR (upper panel). Blots were stripped and reprobed with cyclophilin (cyc) riboprobe (lower panel). RNA size markers (kb) are shown on the left of the blot. (B) Autoradiographs from northern blot studies were analyzed by scanning densitometry and the ratio of signal from the ~5 kb band over the signal from the cyclophilin band was calculated. Mean ratios ± SE are shown. The ratio of ~5 kb transcript signal was significantly decreased in NEDH compared with adrenal medullary tissue (**P < 0.01, n = 5 separate northern blots, Mann–Whitney test). Values obtained from each blot were normalized against the average ratio obtained for Wistar brain.

 
LAR protein level is decreased in NEDH adrenal medulla
Western analysis measuring levels of the LAR ~150 kDa extracellular subunit was performed using antibody directed against the LAR N-terminus (Figure 5A and BGo). Protein extracts were isolated from adrenal cortex and medullary tissue harvested from Wistar and NEDH rats. In adrenal cortex, LAR protein levels were decreased in NEDH samples by an insignificant degree to 87% of those found in Wistar samples. In contrast, in adrenal medulla LAR protein levels in 2–3-month-old rats were decreased significantly in NEDH males to 43 ± 6% and in NEDH females to 60 ± 7% of Wistar levels. This decrease in LAR protein level was consistent with the decrease in levels of LAR transcripts in NEDH medullary tissue (Figure 3Go). Although this decrease tended to be larger in males, the difference between males and females did not reach significance. In adrenal medullary tissue isolated from 12-month-old NEDH rats with histological features of tumor, LAR protein levels were decreased to 50 ± 13% of the levels in medullary tissue from 10–12-month-old Wistar rats and similar to those found in young NEDH rats.



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Fig. 5. Western blot analysis with LAR N-terminal antibody. (A) Pairs of adrenal medullas were combined from 2–3-month-old (three male and three female) and 10–12-month-old (three male and three female) Wistar rats (WIST) for separate protein preparations. Pairs of adrenal medullas were combined from 2–3-month-old male (NEDH-M, six animals) and female (NEDH-F, six animals) NEDH rats for separate protein preparations. Portions of all four adrenal medullas harvested from the two 12 month NEDH rats (NEDH 12Mo) with histological features of tumor were combined to make one tumor protein preparation. Protein (100 µg) extract was resolved by 7.5% SDS gel electrophoresis, transferred to PVDF membrane and probed with affinity-purified LAR N-terminal antibody which identifies the ~150 kDa LAR extracellular subunit. Blots were stripped and reprobed with antibody against the ~42 kDa actin protein. (B) Autoradiograms from western blot analyses were assessed by scanning densitometry. The ratio of signal from LAR protein over the actin band was calculated. Mean ratios ± SE are shown. In 2–3-month-old adrenal cortex tissue, LAR protein levels in Wistar and NEDH samples were similar. In 2–3-month-old adrenal medullary tissue, LAR protein levels in NEDH samples were significantly decreased in both male (**P < 0.01, n = 7 separate western blots, Mann–Whitney test) and female (*P < 0.05, n = 7 separate western blots, Mann–Whitney test) samples. A similar significant decrease was found in 12-month-old NEDH medullary tumor tissue compared with 10–12-month-old Wistar medullary tissue (*P < 0.05, n = 7 separate western blots, Mann–Whitney test).

 
LASE-c splicing is increased in NEDH adrenal medulla and PC12 cells
In the extracellular portion of LAR, the presence or absence of a nine residue segment within the fifth fibronectin domain regulates the affinity of LAR to laminin–nidogen complexes (18). This segment is encoded by an alternatively spliced 27 bp exon, LASE-c (15,16). RT–PCR analysis was used to compare the proportion of LAR transcripts containing the LASE-c insert in Wistar and NEDH adrenal tissue (Figure 6A and BGo). In 2–3-month-old rats, the proportion of LAR transcripts containing LASE-c was increased significantly by 6.2-fold in NEDH males compared with Wistar levels. Similarly, this proportion was increased by 6.4-fold in NEDH females compared with Wistar levels. Proportions in LASE-c splicing were similar between male and female NEDH rats. In 10–12-month-old rats, the proportion of LAR transcripts containing LASE-c was increased markedly by 254- and 88-fold in NEDH rats #274 and #331, respectively compared with proportions found in Wistar 10–12-month-old rats. Only trace levels of LAR transcripts containing LASE-c were detected in adrenal cortical tissue of both Wistar and NEDH rats. These low levels were consistent with previous studies showing that LASE-c splicing occurs preferentially in neural tissues (15).



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Fig. 6. LASE-c alternative splicing in adrenal tissue and PC12 cells. (A) RT–PCR analysis using primers flanking the 27 bp LASE-c insert was performed using 0.5 µg of total RNA isolated from Wistar and NEDH adrenal tissue and PC12 cells. H2O served as a negative control. For rats 2–3 months of age, pairs of adrenal cortexes or medullas were combined from the following three sets of rats: six Wistar (WIST; three males and three females), six NEDH male (NEDH-M) and six NEDH female (NEDH-F). One total RNA preparation was prepared from each pool. For rats 10–12 months of age, pairs of adrenal medullas were combined from two Wistar rats (one male and one female, WIST 10–12Mo) and from the two NEDH rats with adrenal tumors (#274 and #331). One total RNA preparation was prepared from each pool. RT–PCR reactions yielded the expected products of 135 bp (without LASE-c) and 162 bp (with LASE-c). RT–PCR products were electrophoresed in 6% polyacrylamide gels, stained with ethidium bromide and photographed. Only trace levels of LASE-c splicing were detected in adrenal cortical tissue. (B) Photographic negatives were analyzed by scanning densitometry and the ratio of the signal from the upper band (with LASE-c) over the signal from the lower band (without LASE-c) was calculated. The mean ratios ± SE are shown. In 2–3-month-old male and female NEDH medullary tissue, the proportion of LAR transcripts containing LASE-c was significantly higher compared with Wistar samples (***P < 0.001, Mann–Whitney test, n = 9 and n = 10 separate RT–PCR reactions for Wistar and NEDH samples). (C) In 12-month-old NEDH tumor tissue, LASE-c splicing was significantly higher compared with corresponding Wistar samples (***P < 0.001, Mann–Whitney test; Wistar, n = 11 reactions; NEDH #274, n = 9 reactions; NEDH #331, n = 5 reactions). In PC12 cells, LASE-c splicing was significantly increased compared with that found in Wistar 10–12 month samples (***P < 0.0001, Mann–Whitney test, n = 17 reactions for PC12 cells). Note the difference in y-axis scales. The actual value for WIST 10–12Mo is 0.029.

 
In PC12 cells, the increase in LASE-c splicing was even more pronounced (Figure 6CGo). The proportion of LAR transcripts containing LASE-c in PC12 cells was ~80-fold greater than that found in NEDH adrenal medullary tissue of 2–3-month-old rats, and ~500-fold greater than that in the corresponding Wistar adrenal medullary samples. The proportion of LAR transcripts containing LASE-c in PC12 cells was only 1.4- and 4-fold greater than that found in medullary tissue isolated from NEDH rats #274 and #331, respectively, and ~350-fold greater than that measured in 10–12-month-old Wistar medullary tissue. Thus, an increasingly aberrant pattern of LASE-c splicing was found over a continuum progressing from young Wistar tissue to NEDH pre-tumor tissue to NEDH tumor tissue to PC12 pheochromocytoma cells.

Expression of LASE-c-containing LAR protein isoform is increased in NEDH adrenal medulla
An additional series of western analyses was performed using antibody directed against the LASE-c insert recognizing the ~150 kDa LAR extracellular subunit. LASE-c containing LAR protein was present in adrenal cortical and medullary tissues as well as PC12 cells (Figure 7AGo). Inspection of western blots suggested that levels of the LASE-c isoform protein were increased in NEDH compared with Wistar adrenal medulla and that LASE-c protein levels in NEDH adrenal medullary tissue were similar to those found in PC12 cells. Quantitative analysis confirmed that levels of LASE-c containing ~150 kDa LAR protein were increased significantly by 1.8-fold in 2–3 month NEDH compared with 2–3 month Wistar adrenal medulla (Figure 7B and CGo). Moreover, the proportion of LAR protein isoforms containing LASE-c over LAR N-terminal ~150 kDa protein was increased significantly by 4-fold in NEDH compared with Wistar adrenal medulla (Figure 7B and DGo). This 4-fold increase in the proportion of LASE-c-containing protein isoforms was of a similar magnitude to the 6-fold increase in splicing of the LASE-c insert found in LAR transcripts.



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Fig. 7. Western blot analysis with LASE-c antibody. (A and B) Protein extracts from Wistar (WIST) and NEDH adrenal medulla, adrenal cortex, cerebral cortex (CTX, positive control) and PC12 cells were prepared. Pairs of adrenal cortexes or medullas were combined from 2–3-month-old Wistar (three male and three female) and 2–3-month-old NEDH (three male and three female) rats and protein prepared from each pool. Protein extract (100 µg) was resolved by 7.5% SDS gel electrophoresis, transferred to PVDF membrane and hybridized with affinity-purified LASE-c followed by probing with LAR N-terminal antibodies; both identified the ~150 kDa LAR extracellular subunit. As shown in (A), LAR isoforms containing LASE-c were detected in all tissues. (C) Autoradiograms from LAR and LASE-c western blot analyses were assessed by scanning densitometry. The ratio of LASE-c signal over that derived from reprobing blots with actin antibody was calculated. This ratio was significantly increased in NEDH compared with Wistar medullary tissue (*P < 0.05, Mann–Whitney test, n = 5 blots). Mean ratios ± SE are shown. (D) The ratio of signals from LASE-c bands over that from LAR bands was calculated. Mean ratios ± SE are shown. The ratio of LAR protein isoform containing LASE-c over LAR protein were increased significantly by 4-fold in NEDH compared with Wistar adrenal medullary tissue (**P < 0.01, n = 5 separate western blots, Mann–Whitney test).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In human pheochromocytoma, genotyping studies demonstrating loss of heterozygosity have supported the view that these tumors result from decreased or loss of activity of one or more tumor suppressor genes (24). The human LAR gene is located at chromosome 1p32–33, a region associated with multiple human tumors (22). The mapping of human LAR to a locus involved in pheochromocytomas led to the hypothesis that LAR constitutes a tumor suppressor gene involved in these tumors (43).

The high incidence of pheochromocytomas in the NEDH line of Wistar rats provides a useful rodent model for identifying aberrations in gene expression contributing to the formation of these tumors. The present studies demonstrate that LAR mRNA and protein levels are decreased in NEDH adrenal medullary tissue harvested at 2–3 months of age, a period well before early stages of tumors become evident. A similar degree of decreased LAR protein levels was found in NEDH medullary tumor tissue. Pre-tumor adrenal medullary tissue also demonstrated increased splicing of the LASE-c insert in LAR transcripts and a corresponding increase in the proportion of LAR protein isoforms containing the LASE-c domain. This aberrant pattern of LAR extracellular domain splicing was dramatically increased by some 80–250-fold in tumor tissue and up to 500-fold in PC12 cells. The findings of a loss of the ~5 kb LAR mRNA isoform in PC12 cells along with decreased LAR protein levels and increasingly aberrant LASE-c splicing in NEDH pre-tumor and tumor tissue suggests a progressive continuum of altered LAR expression and splicing moving from Wistar medullary tissue to NEDH tissue predisposed to pheochromoctyoma formation to actual NEDH-derived pheochromocytoma cells. It is important to note, however, that since radiation of NEDH rats was used to generate the P259 tumor from which the PC12 cell line was derived (29,31); it is likely that mutations in multiple genes contribute to the overall PC12 cell phenotype. The present findings point to the gene encoding LAR or genes involved in regulation of LAR expression and/or splicing as candidate genes contributing to formation of rat pheochromocytomas.

Previous studies demonstrating decreased expression or altered splicing of PTPs in tissues with well-established tumors raised the possibility that changes in PTP levels or isoforms might contribute to tumor formation (26). Since studies of PTP expression in the context of tumors have been largely limited to assessment of tumor tissue itself, it is possible that these changes might be strictly a result of rather than a mechanism contributing to tumor phenotype. The finding in the present study of altered LAR expression and a tumor-associated pattern of aberrant splicing present in tissue predisposed to tumor formation, or in very early stages, supports the hypothesis that changes in LAR function might contribute to transformation. The observation that LAR isoforms containing the LASE-c insert have decreased affinity to laminin–nidogen complexes (18) raises the possibility that the increases in LASE-c splicing in NEDH medullary tissue, tumor tissue or PC12 cells might lead to altered interactions between pheochromocytoma cells and the surrounding extracellular matrix.

Whereas the present study demonstrates an important association between changes in LAR expression and splicing in medullary tissue predisposed to pheochromocytoma, it does not demonstrate a cause-and-effect relationship. The discovery of this association has encouraged ongoing studies in our laboratory in which PC12 cells were stably transected with null-vector and LAR antisense transgenes. LAR antisense PC12 cell clones have decreased levels of LAR protein and significantly increased proliferation rates (21). These functional studies further support the hypothesis that LAR regulates proliferation of adrenal medullary cells. The present study will also encourage studies of pheochromocytoma predilection using LAR null-mutant mice alone and in combination with other mouse models of pheochromocytomas (44).


    Acknowledgments
 
We thank Drs Ben Yen and Carolyn Montgomery, VAMC/UCSF Department of Anatomic Pathology for assistance in reviewing adrenal gland histology. This study was supported by the American Cancer Society (F.M.L.), a Beeson Award from the American Federation for Aging Research (F.M.L.) and a Veterans Administration Merit Review (F.M.L.).


    Notes
 
2 To whom correspondence should be addressedEmail: lfm{at}itsa.ucsf.edu

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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 

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Received March 18, 1999; revised October 5, 1999; accepted October 14, 1999.