Atria1 natriuretic peptide synthesis in atria1 tumors of transgenic mice DAVID G. GARDNER, MARIA J. F. CAMARGO, RICHARD R. BEHRINGER, RALPH L. BRINSTER, JOHN D. BAXTER, STEVEN A. ATLAS, JOHN H. LARAGH, AND CHRISTIAN F. DESCHEPPER Departments of Medicine and Physiology and Metabolic Research Unit, University of California, San Francisco, California 94143; Hypertension Center, Cornell University Medical College, New York, New York 10021; Laboratory of Reproductive Physiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Gardner, David G., Maria J. F. Camargo, Richard R. Behringer, Ralph L. Brinster, John D. Baxter, Steven A. Atlas, John H. Laragh, and Christian F. Deschepper.
Atria1 natriuretic peptide synthesis in atria1 tumors of transgenie mice. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E524E531,1992.-Transgenic mice harboring a chimeric gene linking mouse protamine 1 W-flanking sequence to the coding sequence of the simian virus 40 T-antigen develop spontaneous rhabdomyosarcomas of the right atria. The presence of the tumors is accompanied by dramatic elevations in plasma atria1 natriuretic peptide (ANP) immunoreactivity (1,698 & 993 vs. 60 & 18 fmol/ml for controls) and hematocrit (56 t 8 vs. 51 t 2 for controls). The immunoreactive ANP (irANP) present in the tumors is similar in size to irANP found in normal mouse atria. ANP mRNA transcripts present in the tumors also appear to be very similar in overall size and 5’-termini to those produced in normal cardiac tissue. Microscopically, the tumors are composed of a disorganized array of densely packed abnormal-appearing cells. Immunocytochemistry and in situ hybridization analysis reveal considerable heterogeneity in ANP gene expression. ANP peptide and mRNA are detectable throughout the parenchyma of the tumors, but absolute levels of expression vary widely among different cells in the population. These tumors represent a potentially valuable model for the study of inappropriate ANP secretion and may provide a tissue source for the development of an ANP-producing atria1 cell line. rhabdomyosarcoma; protamine gene expression; in situ hybridization THEATRIAL NATRIURETIC PEPTIDE (ANP)is ahormone that is synthesized and secreted predominantly by the cardiac atria (1). Its natriuretic and vasorelaxant properties, as well as its well-described antagonism of the renin-angiotensin-aldosterone axis (1, 15), suggest that it represents a naturally occurring antihypertensive peptide that opposes stimuli of volume expansion and increased arterial blood pressure. Recently, it was reported (2) that introduction of a chimeric gene, linking the Y-flanking sequences (5’FS) of a mouse protamine 1 gene (mP1) to the coding sequence of the simian virus 40 (SV-40) T-antigen, into transgenic mice resulted in the seemingly fortuitous development of atria1 rhabdomyosarcomas as well as bilateral osteosarcomas of the petrous portion of the temporal bone. In the current study, we have determined that the capacity for ANP gene expression remains intact in a subpopulation of the transformed atria1 cardiocytes in these tumors and that this expression is accompanied by elevated levels of ANP in the circulating plasma. E524
0193-1849/92
$2.00
Copyright
MATERIALS AND METHODS Materials. Restriction and modification enzymes were obtained from either Bethesda Research Laboratories or Boehringer Mannheim. [a-32P]dCTP (X,000 Ci/mmol) and [cY-~~P]dATP (~400 Ci/mmol) were obtained from Amersham; [T-~~P]ATP (~3,000 Ci/mmol) was purchased from ICN. Antirat (r) ANP antisera were purchased from either Research and Diagnostic (Emeryville, CA) or Peninsula Laboratories (Belmont, CA). Tissue preparation. mPl-SV-40 transgenic mice were originally generated by microinjection of the chimeric gene construct into the pronuclei of C57BL/6 x SJLF2 fertilized eggs. Characterization of these lines has been described (2). The lines were maintained by breeding transgenic animals with C57BL/6 x SJLF, nontransgenic controls. C57BL/6 X SJLF, age-matched mice served as controls for this study, Tumorbearing animals were killed at -10-20 wk of age, and tumors, dissected free of attatched normal myocardium, and ventricular tissue were collected, quick-frozen in liquid nitrogen, and stored at -70°C until processed. Normal atria and ventricles were also collected from nontransgenic mice and pooled for analyses. Hematocrit was measured on freshly collected blood after centrifugation in a bench-top hematocrit centrifuge. For measurement of plasma ANP, trunk blood from freshly killed animals was collected in EDTA, and plasma was obtained by centrifugation at 500 g at 4OC. Preparation and analysis of RNA. Total RNA was extracted from the atria1 tumors or normal cardiac tissue using the method of Cathala et al. (6). RNA was size fractionated on a 1% agarose gel and blot hybridized to an rANP cDNA probe (29) labeled by nick translation [ -lo8 counts. min”(cpm). pg-‘I. Blot hybridizations were carried out as described previously (26), except that prehybridization buffer was used for both the prehybridization and the hybridization steps, the temperature of the hybridization was 32”C, and the wash was in x0.1 standard sodium citrate (0.15 M NaC1/0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate at 23°C. The latter modifications were introduced to optimize the association of the heterologous rANP cDNA probe with the mouse ANP transcript. Autoradiograms were obtained for individual blots, and levels of ANP transcripts were approximated using scanning laser densitometry. Primer extension analysis was carried out as described previously (12) using a 23-nucleotide primer corresponding to residues 545-569 of the complementary strand of the mouse ANP gene (20). This sequence reads 5’-GGGTCGGGGCACGATCTGATGTT-3’. Products were size fractionated on a denaturing 6% polyacrylamide gel. S1 nuclease protection analysis was carried out as described previously (12) using a 5’-end labeled 347-base pair (bp) Hind III-BgZ II fragment, which spans the transcription start site on the mouse ANP gene. The predicted size of the fragment
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING
TUMORS
protecting the &end of the mouse ANP transcript was 194 nucleotides. DNA sequencing. Chain-termination DNA sequencing was carried out on the double-stranded mPl-SV-40 template using a 22nucleotide primer corresponding to positions +80 to +lOl (relative to the transcription start site) in the complementary strand of the SV-40 early-region sequences (11). Sequence was also generated on the alternate strand using a 26-nucleotide primer corresponding to residues +45 to +70 in the mP1 5’FS (18). Each primer provided an identical double-stranded sequence across the mPl-SV-40 junction region. Tissue extract preparation and analysis for ANP immunoreactiuity. ANP was extracted from atria1 tumors and normal atria1 tissue as described by DeBold and Flynn (8). Briefly, frozen tissue was homogenized in 10 vol of an ice-cold extraction solution [ 1 M acetic acid, 1 M HCl, 1% NaCl, pepstatin A (1 mg/l), and phenylmethylsulfonyl fluoride (1 mg/g tissue)] using a polytron PT 35 for 60 s at 50% maximum power. The homogenate was centrifuged at 10,000 g for 30 min. The pellet was reextracted with 5 vol of extraction solution, and the supernatants were combined and frozen at -40°C until highperformance liquid chromatography (HPLC) analysis. Immunoreactive ANP (irANP) in plasma, atria1 tumor, and normal atria1 tissue was measured by a direct radioimmunoassay (RIA) as described (7) using an antiserum to human (h) ANP-(99-126), with the same peptide used as standard (Peninsula Laboratories) and trace (Amersham). HPLC fractions and tissue extracts were assayed directly; plasma samples were assayed after extraction on Cls Sep-Pak cartridges (Waters) as previously described (7). Tissue irANP levels reported in Table 1 were measured on small aliquots of the guanidinium monothiocyanate (GSCN) suspension used for isolation of RNA (see above). GSCN had no effect on the RIA at concentrations present in the assay mixture. Samples were resuspended in 100 rug assay buffer (0.1 M NaCl, 0.05 M sodium phosphate, pH 7.2, 0.1% bovine serum albumin, 0.01% Triton X-100, 0.01% NaN& and were assayed using a rabbit anti-hANP antiserum (Research and Diagnostics). The latter antibody displayed 30% cross-reactivity with human pro-ANP; the Peninsula antibody was 40% cross-reactive with the prohormone. Both antibodies employed have 100% cross-reactivity to rANP; the latter is identical in sequence to mouse ANP (20,27). Extracts of plasma, atria1 tumor, and normal mouse atria were analyzed by reverse-phase HPLC using a Cls PBondapak Table 1. Immunoreactiue of transgenic mice Sample
ANP levels in tumors irANP, pg ANP/pg soluble protein
Pooled normal atria 15.18 Pooled normal ventricles 0.25 B75 Tumor 7.01 Ventricle 0.36 B77 Tumor 15.57 Ventricle 0.36 B79 Tumor 3.05 Ventricle 0.21 B80 Tumor 15.33 Soluble protein was prepared from homogenized tissue (i.e., tumor or normal ventricular tissue from same animal) as described in MATERIALS AND METHODS and was assayed for immunoreactive atria1 natriuretic peptide (irANP) using a Research and Diagnostic anti-rat ANP antisera. See legend to Fig. 1 for identification of individual transgenic animals (B75, B77, B79, and B80).
IN TRANSGENIC
MICE
E525
column (Waters) developed at a flow rate of 1 ml/min with a gradient of lo-60% acetonitrile in 0.1% trifluoroacetic acid over 50 min. One-minute fractions were collected; eluates were dried in a Speed Vat evaporator and reconstituted in buffer immediately before RIA. After each injection, the retention time of 1251-labeled hANP was determined as a reference marker. Differences were documented by analysis of variance and the Newman-Keuls test for significance or by Student’s t test. Immunocytochemistry and in situ hybridization histochemistry. Excised hearts bearing the atria1 tumors were placed directly into Bouin’s sublimate, fixed, sectioned, and processed as described previously (12). Immunostaining was carried out using a rabbit anti-rANP antiserum (AP l-2) raised against thyroglobulin-coupled rANP. The titer of the antibody was 1:60,000 as determined by binding of 12’1-labeledrANP to serial dilutions of the antiserum. The antiserum was used at a l:l,OOO dilution for the immunocytochemical studies. As a negative control, the antiserum, at a l:l,OOO dilution, was preabsorbed to the rANP peptide (final concentration 10 pg/ml) before incubation with the atria1 section. This resulted in the elimination of immunostaining in the section (data not shown). For the hybridization studies, atria1 tumors, normal atria, and ventricles were embedded in OCT compound and frozen immediately in a methanol-dry ice bath. Subsequent preparation and hybridization to a [3H]cRNA probe for rANP were carried out as described previously (9). Sections were exposed for 3-4 wk before development of the photographic emulsion. Hybridization with a [3H]mRNA rANP probe revealed only background levels of hybridization in each of the tissues examined (data not shown). RESULTS
Plasma ANP levels in five transgenic mice bearing atria1 tumors averaged 1,698 t 993 (SD) fmol/ml. These levels were -30-fold higher than levels in control mice (60 t 18 fmol/ml; P < 0.01 ) of the same strain maintained on the same diet. Measurements of hematocrit, as an assessment of ANP bioactivity in vivo (1, 15), were made on transgene-bearing mice as well as a similar number of their nontransgenic litter mates. Hematocrits were, in general, elevated in the transgenic group [56 t (SD) 8 vs. 51 t 2 in the nontransgenic animals; P < 0.01; n = 26 for the former and 25 for the latter group], raising the possibility that the elevations in ANP promote significant alterations in volume homeostasis. Given the large size of these tumors, elevations of plasma ANP in these mice could arise either directly through secretion from the atria1 tumor cells or from tumor-related obstruction to atria1 outflow with elevated intra-atria1 pressure and increased ANP release from the neighboring atria1 tissue. To address this issue, we examined the tumor tissue for the presence of the ANP peptide as well as the ANP mRNA transcript. As shown in Fig. 1, the atria1 tumors possess significant quantities of ANP mRNA, which is of the same size and 40-15% the abundance of that produced in the normal cardiac atria. The cardiac ventricles, on the other hand, contained only small amounts of the ANP mRNA, a finding similar to that reported earlier for rats (12). The high levels of ANP mRNA were accompanied by high levels of irANP protein in the tumors (Table 1). Extracted plasmas from the transgenic and control animals were subjected to HPLC analysis. As shown in
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
E526
ANP-PRODUCING
TUMORS
IN
TRANSGENIC
MICE
123456789
Fig. 1. Blot-hybridization analysis of atria1 natriuretic peptide (ANP) transcripts in RNA from atria1 tumors. Total RNA from pooled normal mouse atria (3 fig, lane l), pooled normal mouse ventricle (30 pg, Lane 2), ventricle from mouse B75 (20 pg, lane 3), atria1 tumor from B75 (20 pg, lane 4), ventricle from B77 (20 pg, lane 5), atria1 tumor from B77 (20 pg, lane 6) ventricle from B79 (20 pg, lane 7), atria1 tumor from B79 (20 Fg, lane 8), or atria1 tumor from B80 (20 wg, lane 9) was size fractionated, blotted to nitrocellulose filters, and hybridized to rat ANP cDNA probe. Transcripts were -950 nucleotides in size based on comparison with DNA markers run in parallel. Subsequent reprobe of blot with radiolabeled actin cDNA revealed roughly equivalent levels of actin transcript in each tumor RNA lane. Animal B75, 18-wk-old male from 1738-3 line; B77, 16-wk-old male from 1736-l line; B79, 16-wk-old male from 1738-4 line; BSO, 15-wk-old male from 1736-8 line.
Fig. 2 the major form(s) of irANP in the plasma of the normal (Fig. 20) and transgenic mice (Fig. 2B) migrated very close to the position of hANP-(99-126), eluting l2 min ahead of the ‘251-labeled standard. On the other hand, extracts from either normal mouse atria (Fig. 2C) or the mouse atria1 tumors (Fig. 2A) contained large amounts of late-eluting immunoreactivity, corresponding to the ANP precursor [ANP-(l-126); retention time 3538 min] in addition to mature ANP and a variety of intermediate forms (5). Broadening of the initial portion of the immunoreactive peaks presumably represents
4
lz51hANP 4 P I/ I/
NH2-terminally degraded metabolites that are detected by the antibody. To assure that the observed expression was being driven from the native ANP gene promoter, we carried out primer extension and nuclease Si protection analyses on RNA from the atria1 tumors. As shown in Fig. 3A, primer extension produced products 82-86 nucleotides in length. This corresponded to 5’-termini 23-27 bp downstream from the TATA box sequence in the mouse ANP gene (20), which is believed to dictate the initiation of the dominant ANP transcript. This start was also rf;000
4000 Cl 2000
60
-I
18
Retention
time
6 2 3 =I
Fig. 2. Reverse-phase high-performance liquid chromatography of extracts of plasma and atria1 tissue from tumorbearing transgenic mice and normal control mice. Plasma or tissue extracts injected were equivalent to 12 mg atria1 tumor (A), 30 ~1 transgenic mouse plasma (B), 215 fig normal mouse atria (C), and 190 ~1 normal mouse plasma (D). Broken lines, elution pattern of iZ51labeled human ANP (hANP; 10,000 counts/min) injected after each experiment (scale shown at right). Major early eluting peaks of immunoreactive ANP (irANP) had retention time (18-20 min) consistent with that of ANP-(99-126); as expected, elution of ‘251-labeled standard was delayed by l-2 min.
(mln)
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING
12
3
4
5
TUMORS
IN
TRANSGENIC
MICE
6
-242 >
-190
E527
Fig. 3. Identification of 5’-termini of mouse ANP transcripts in atria1 tumor. A: primer extension analysis of total RNA from normal mouse ventricle (20 pg, lane 1 ), normal atria (1 rg, lane 2) or atria1 tumor from B75 (20 pg, lane 3), B77 (20 pg, lane 4), B79 (20 pg, lane 5), and B80 (20 rg, lane 6). Arrow, position of dominant mouse ANP transcript. B: nuclease S, protection of total RNA from normal atria (3 pg, lane I), normal ventricle (20 pg, lane 2) or atria1 tumors from B75 (30 pg, lane 3), B77 (30 wg, lane 4), and B80 (30 gg lane 5). Numbers to right represent size in base pairs of DNA markers run in parallel.
-147
identified in RNA from normal atria1 and ventricular tissue (Fig. 3A). Nuclease Si protection was carried out using a 347-bp Hind III-Bgl II fragment that spans the 5’-end of the mouse ANP gene. This probe (Fig. 3B) protected a fragment -195 nucleotides in length, with RNA from atrial, ventricular, or tumor tissue, mapping the start site to a position overlying that demonstrated by primer extension above. Finally, to confirm that expression of the ANP gene was carried out within the tumor cells, we employed immunocytochemistry and in situ hybridization histochemistry to localize ANP gene expression at a cellular level. Immunocytochemical analysis for irANP (Fig. 4A) in the normal atria revealed a fairly homogenous pattern of immunostaining with a dense perinuclear concentration of the peptide. In the case of the tumor section, the staining pattern was considerably more heterogenous (Fig. 4C). Individual cells scattered throughout the tumor parenchyma stained to varying degrees for ANP immunoreactivity. Positive cells tended to be clustered together in a swirling pattern coursing among cells displaying much lower, or nonexistent levels of irANP. Hybridization of normal mouse atria1 sections with an rANP [3H]cRNA probe revealed a relatively intense signal that spread across the full thickness of the atria1 wall (Fig. 4B), mirroring the distribution of immunoreactivity. A similar pattern of expression has been reported previously in the l-day-old mouse heart (30). The signal was very low to absent in the mouse ventricle (data not shown). Hybridization of the [3H]cRNA to sections of the atria1 tumor (Fig. 40) revealed a much more heterogenous pattern with areas of high-intensity signal superimposed on a lower-intensity background. This signal was in general lower than that seen in “normal” atria1 tissue (see Fig. 4C) but considerably higher than that found in the cardiac ventricle (data not shown). The regions displaying the most intense hybridization activity followed a swirling pattern that resembled that described above for ANP immunoreactivity. DISCUSSION
As mentioned above, the expression of the mPl-SV40 transgene results in the seemingly fortuitous appear-
ance of atria1 and temporal bone tumors (2). The expression of T-antigen was more pronounced in the atria1 tumors than it was in round spermatids of the testes, cells that usually employ the mP1 promoter with a high degree of efficiency (18). It is noteworthy that testicular tumors were absent. Previous investigations have provided no evidence suggesting increased expression of either the endogenous mP1 gene (2, 14) or an mP1 transgene (18) in cardiac tissues. In addition, numerous studies employing a variety of SV-40 T-antigen-containing transgenes have failed to provide evidence for selective expression of these sequences in cardiac tissue (17). Thus it appears that expression of the mPl-SV-40 gene results from the utilization of a tissue-specific element(s) that may have been created fortuitously by linking the SV-40 and protamine gene sequences. Such novel expression patterns have been noted previously in transgenic mice harboring chimeric metallothionein-growth hormone fusion genes (19, 24). This could result either from the synergistic interaction of normally quiescent sequences present in each of these genes or alternatively from the de novo generation of a unique element at the point of fusion of these two genes. To examine the latter possibility, DNA sequence analysis was carried out across the mPl-SV-40 junction region. This sequence is presented in Fig. 5. We have compared the sequence of the junction region with that of a DNA segment from the hANP gene (i.e., positions -385 to -339), which we have recently shown by a combination of functional and gel mobility-shift analyses to be important in regulating expression of that gene (28). As shown in Fig. 5, a region of shared homology can be identified. When aligned for maximal overlap, the two sequences are homologous at 19 of 28 positions. In the case of the mPl-SV-40 construction, the homologous sequence is derived entirely from the mP1 gene and polylinker-adapter sequence. These findings raise the possibility that linkage of the protamine and SV-40 gene sequences through a synthetic adapter-linker may have contributed, in part, to the creation of a cis-acting regulatory element that, in the context of the surrounding transgene sequence, allows for increased expression of the viral T-antigen and consequent expression of the transformed phenotype in
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
E528
ANP-PRODUCING
TUMORS
IN
TRANSGENIC
MICE
Fig. 4. A: immunocytochemistry of ANP on section of normal mouse atrium. No hematoxylin counterstaining. Immunostaining is spread homogeneously across section. Particularly dense concentrations of staining are noted in a perinuclear distribution. Magnification ~63. Bar = 75 pm. B: in situ hybridization histochemistry of section of normal mouse atrium, using rat ANP [3H]cRNA probe; photographed in darkfield illumination. Cell nuclei were counterstained with hematoxylin. Note that a small rim of connective tissue with more densely packed nuclei can be seen on right corner (see arrow) where no signal is present. No signal was observed when a rat ANP [3H]mRNA probe was used on adjacent sections (not shown). Magnification x40. Bar = 100 pm. C: immunocytochemistry of ANP on transgenic mouse atria1 tumor section. No hematoxylin counterstaining. Positive immunoreactivity appears as dark deposits of diaminobenzidine (see arrows for example of immunopositive cells). Appearance of tumor section is that of swirls of spindle-shaped cells separating clusters of more densely packed cells. Although ANP immunoreactivity was detected throughout tumor, it was especially dense in these cellular swirls where ratio of cytoplasmic to nuclear area was highest. Magnification x40. Bar = 100 pm. D: in situ hybridization histochemistry on transgenic mouse atria1 tumor section using rANP [3H]cRNA probe. Cell nuclei were counterstained with hematoxylin. Distribution of signal in atria1 tumor was similar to what was observed with immunocytochemistry [i.e., while scattered throughout tumor, signal was more intense in those areas harboring swirls of spindle-shaped cells (see arrows)].
atria1 cells. However, the mechanism is likely to be more complicated than this. Field (10) recently reported that introduction of a chimeric construction linking 500 bp of 5’FS from the hANP gene, a segment that includes the tissue-specific element (28) alluded to above, to the SV-40 T-antigen coding sequences resulted in the generation of grossly enlarged right atria. Pathologically these atria were characterized by hyperplasia rather than the rhabdomyosarcomas seen with the mPl-SV-40 transgene. In addition, Stewart et al. (23) recently reported the generation of transgenic mice bearing the regulatory sequences of the mP2 gene, which bears a high degree of sequence homology to mP1 (13), linked to the coding sequences of the SV-40 T-antigen. In that case the transgene was expressed in cardiac cells, but tumorigenesis was not reported (23). Thus there are in all likelihood additional factors present in the mPl-SV-40 construct
that are responsible for the complete manifestation of the malignant phenotype. Of additional interest is the finding that both the hANF-SV-40 Tag gene of Field (10) and the mPl-SV-40 Tag gene described by Behringer et al. (2) appear to express the neoplastic phenotype preferentially in the right vs. left atria. This could imply selective chamber-specific utilization of transcriptional regulatory signals in the chimeric genes. Because the endogenous ANP gene shows no such preferential expression, these signals are likely to involve elements outside the ANP promoter or the homologous element identified in the mPl-SV-40 Tag fusion gene (see above). An alternate possibility is that expression of these genes alone may be necessary but not sufficient for malignant transformation. Exposure to a second growth-promoting factor, with a topographically limited distribution of expression, might provide the requisite stimulus for dem-
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING
TUMORS
IN TRANSGENIC
E529
MICE -337
+81
+123 n I
mpl
n n
SFS
Adapter/Bgl II sv40 Linker Fig. 5. Sequence of mP1-SV-40 fusion region. Sequence from sense strands of hANP 5’FS and fusion region of mPlSW40 construction are presented. hANP sequence is numbered relative to major transcription start site in hANP gene. mP1 sequence is numbered relative to mP1 start site, which appears to be employed in chimeric gene (3). Contributions of mP1 YFS, synthetic linker-adapter, and SV-40 T-antigen coding sequences are delineated below mPl-W-40 sequence. Sequences have been aligned for maximal overlap.- Regions with 2 or more sequential homologous bases are shaded.
onstration of the full neoplastic phenotype. The latter hypothesis is supported by the studies of Field (lo), alluded to above, in that he found evidence for Tag expression in both the right and left atria despite the strong predilection for neoplasia on the right side. The extreme elevations in plasma ANP noted in several of the mice were sufficient to lead to a gross disturbance in volume regulation as reflected by the elevations in hematocrit. Steinhelper et al. (21) recently reported on the generation of a mouse line harboring a transgene linking the mouse transthyretin promoter to the structural gene for ANP. These mice produced large amounts of pro-ANP in the liver. The latter finding was expected since the liver avidly expresses th ,e endogenous transthyretin gene. PI .asma ANP leve 1s in these animals were elevated significantly, and, most importantly, they displayed a number of physiological characteristics suggestive of chronic ANP excess. These findings, similar to those described in the present study, suggest that chronic endogenous hypersecretion of ANP can result in sustained physiological perturbations in cardiovascular homeostasis. Although we cannot rigorously exclude a contribution from juxtapositioned normal or near-normal atria1 tissue to the elevation in circulating plasma ANP levels, the presence of ANP transcripts and ANP immunoreactivity within the tumor mass strongly suggests that the elevated plasma levels result, at least in part, from synthesis of ANP within the tumor itself. In addition, preliminary studies indicate that cultured explants of tumor tissue continue to secrete irANP for several months after removal from the host animals (S. Hauschka and C. Gartside, personal communication), supporting the hypothesis that the elevated plasma levels derive from tumor production of ANP. ANP mRNA levels in most of the tumors were somewhat less than those found in normal atria1 tissue (Fig. 1) but considerably higher than that found in either pooled ventricular tissue from normal animals or ventricular tissue taken from the same transgenie mice. Given the large size of these tumors relative to the normal atria, it is likely that tumor-directed ANP synthesis represents a significant fraction of total ANP production in these mice. One exception was the tumor RNA sample shown in lane 8 of Fig. 1, where ANP transcripts were not seen. ANP mRNA was detectable in the more sensitive primer extension analysis (Fig. 3A, lane 5) but was still considerably less than that found in
the other tumor RNA preparations. This does not reflect degradation of the RNA or methodological problems with the analysis, since rehybridization of the blot to a radiolabeled human actin cDNA probe demonstrated roughly equivalent levels of actin transcript in each of the tumor RNA preparations (data not shown). This same tumor demonstrated the lowest level of ANP immunoreactivity in the group, although it was still considerably higher than ventricular levels. These findings suggest that the heterogeneity seen in plasma ANP levels may result not only from differences in individual tumor mass within the transgenic population but also from intrinsic variability among the tumors in terms of their capacity to express the ANP gene and synthesize the encoded protein. Variability in the latter case may reflect cellular heterogeneity in the levels of mANP gene expression which, based on the immunocytochemical and in situ hybridization studies, appears to vary considerably among the different cells in the population. The reasons underlying this heterogeneity given a common genotypic background among the cells remains unknown. The overall size and 5’-termini of the tumor transcripts appear to be similar if not identical to those of the normal mouse atria, indicating that expression of the ANP gene is being driven from its native promoter. In addition, extracts of tumor, like normal atria, contained large amounts of pro-ANP and other high-molecularweight forms, whereas the mature hormone predominated in plasma (Fig. 2). These findings imply that introduction of the transgene has not resulted in gross alterations in ANP gene expression per se but rather that the transformation has resulted in an overproliferation of ANP-producing atria1 cells. A fraction of these transformed atriocytes, at least at the time of tumor resection, appear to have retained the ability to express the ANP gene at a modestly reduced level and to process the prohormone product appropriately. Interestingly, a recent case report of a patient with multicentric rhabdomyoma of the heart (25) described the presence of ANP immunoreactivity in the atria1 but not ventricular lesions, a finding that supports the hypothesis put forth above (i.e., retention of the atria1 phenotype in the neoplastic cells). The implications of the availability of these tumors are numerous. Selective generation of tumors through the transgene approach has been shown to provide a useful source of transformed cell lines retaining charac-
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
E530
ANP-PRODUCING
TUMORS
teristics of the tissue of origin (16). This has particular importance in terms of the atria1 myocytes since no continuous culture line presently exists. Employing transplanted hyperplastic atria1 tissue fragments from their hANF-SV-40 Tag-bearing transgenic mice as a tissue source, Steinhelper et al. (22) succeeded in establishing atria1 myocyte cultures capable of maintaining a differentiated phenotype and replicating in vitro; however, these cells remain difficult to passage continuously in culture. Thus a transformed ANP-producing atria1 rhabdomyosarcoma cell line may offer an additional advantage in terms of facilitating studies of ANP secretion and/or biosynthesis in vitro. In addition, these tumors may represent a viable model of inappropriate ANP hypersecretion, with the potential for providing valuable information regarding the long-term effects of high circulating levels of ANP on volume status and fluid and electrolyte homeostasis in a tumor-bearing animal. Such information could take on clinical importance, given the recent demonstration of ectopic ANP production in a number of human lung carcinomas (3). We thank Bill Pugh for preparation of the manuscript. We also thank J. Wu for assistance with the sequence analysis and B. Hedges, K. Nguyen, and K. Nakamura for technical assistance. We are also grateful to J. Peschon for providing us with the chimeric plasmid (mPlSV-40) and SV-40 oligonucleotide primer employed in DNA sequencing studies, to L. Field for providing the mouse ANP probe used in the nuclease S1 protection study, and to L. Kedes for providing the human actin cDNA. This work was supported by National Heart, Lung, and Blood Institute Grants HL-35753, HL-38774, and HL-18323SCR and by a Grant-in-Aid from the American Heart Association, with partial support from the Redwood Empire Chapter. D. G. Gardner is an Established Investigator for the American Heart Association. Address for reprint requests: D. G. Gardner, Dept. of Medicine, Metabolic Research Unit, Univ. of California, San Francisco, CA 94143. Received 3 September 1991; accepted in final form 13 November 1991. REFERENCES 1. Baxter, J, D., J. A. Lewicki, and D. G. Gardner. Atria1 natriuretic peptide. Biotechnology 6: 529-546, 1988. 2. Behringer, R. R., J. J. Peschon, A. Messing, C. L. Gartside, S. D. Hauschka, R. D. Palmiter, and R. L. Brinster. Heart and bone tumors in transgenic mice. Proc. A&l. Acud. Sci. USA 85: 2648-2652,1988. R. I. Linnoila, M. J. Birrer, A. F. 3. Bliss, D. P., J. F. Battey, Gazdar, and B. E. Johnson. Expression of the atria1 natriuretic factor gene in small cell lung cancer tumors and tumor cell lines. J. Nutl. Cancer Inst. 82: 305-310,199O. R. L., H. Y. Chen, M. E. Trumbauer, M. K. Yagle, 4, Brinster, and R. D. Palmiter. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Nutl. Acud. Sci. USA 82: 4438-4442,1985. M. J. F., J. H. Laragh, and S. A. Atlas. Character5. Camargo, ization of atria1 natriuretic factor released by cultured human atria1 myocytes, In: Biologically Active Atria1 Peptides, edited by B. M. Brenner and J. H. Laragh. New York: Raven, 1987, vol. I, p. 2l26. G., J. F. Savouret, B. L. Mendez, M. Karin, J. A. 6. Cathala, Martial, and J. D. Baxter. A method for isolation of intact translationally active ribonucleic acid. DNA NY 2: 329-335, 1983. Cody, R. J., S. A. Atlas, J. H. Laragh, Covit, K. S. Ryman, A. Shaknovitch, F. Camargo, R. M. Scarborough, and
S. H. Kubo, K. Pandolfino, J. A. Lewicki.
A. B. M. J.
Atria1 natriuretic factor in normal subjects and heart failure patients. J. Clin. Invest. 78: 1362-1374, 1986. DeBold, A. J., and T. G. Flynn. Cardionatrin I-a novel peptide
IN TRANSGENIC
MICE
with potent diuretic and natriuretic 302,1983. 9. Deschepper,
properties. Life Sci. 33: 297-
C. F., S. J. Mellon, J. Jen, and Y. F. Lau.
T. L.
Reudelhuber,
D. G.
In situ hybridization histochemistry on mRNA in endocrine tissues. In: Selected Topics in lMoZecuZurEndocrinology,. edited by Y. F. Lau. New York: Oxford Univ. Press, 1987, p. 31-41. T antigen transgenes 10. Field, L. J. Atria1 natriuretic factor-SV40 produce tumors and cardiac arrhythmias in mice. Science Wash. DC 239: 1029-1033,1988. 11. Fiers, W., R. Contreras, G. Haegeman, R. Rogiers, A. Van Gardner,
de Voorde, G. Volckaert,
H. Van Heuverswyn, and M. Ysebaert.
H. Swyn,
J. Herreweghe,
Complete nucleotide sequence of SV40 DNA. Nature Land. 273: 113-120,1978. 12. Gardner,
D. G., C. F. Deschepper, W. F. Ganong, S. Hane, J. D. Baxter, and J. Lewicki. Extra-atrial expres-
J. Fiddes,
sion of the gene for atria1 natriuretic USA 84: 2175-2179,1986. 13. Johnson,
P. A., J. J. Peschon, B. Hecht. Sequence
factor. Proc. Nutl. Acud. Sci.
P. C. Yelick,
R. D. Palmiter,
homologies in the mouse protamine 1 and 2 genes. Biochim. Biophys. Actu 950: 45-53,1988. 14. Kleene, K. C., R. J. Distel, and N. B. Hecht. cDNA clones encoding cytoplasmic poly(A)+ RNAs which first appear at detectable levels in haploid phase of spermatogenesis in the mouse. Deu. Biol. 98: 455-464, 1983. 15. Maack, T., M. J. F. Camargo, H. D. Kleinert, J. H. Laragh, and S. A. Atlas. Atria1 natriuretic factor: structural and functional properties. Kidney Int. 27: 607-615, 1985. 16. Ornitz, D. M., R. E. Hammer, A. Messing, R. D. Palmiter, and R. L. Brinster. Pancreatic neoplasia induced by SV40 T antigen expression in acinar cells of transgenic mice. Science Wash. DC 238: 188-193,1987. 17. Palmiter, R. D., H. Y. Chen, A. Messing, and R. L. Brinster. SV40 enhancer and large-T antigen are instrumental in development of choroid plexus tumors in transgenic mice. Nature Land. 316: 457-460,1985. 18. Peschon, J. J., R. R. Behringer, R. L. Brinster, and R. D. Palmiter. Spermatid-specific expression of protamine 1 in transgenie mice. Proc. Nutl. Ad. Sci. USA 84: 5316-5319,1987. 19. Russo, A. F., E. B. Crenshaw, S. A. Lira, D. M. Simmons, L. W. Swanson, and M. G. Rosenfeld. Neuronal expression of chimeric genes in transgenic mice. Neuron 1: 311-320, 1988. 20. Seidman, C., K. Bloch, K. Klein, J. Smith, and J. Seidman. Nucleotide sequences of the human and mouse atria1 natriuretic factor genes. Science Wash. DC 226: 1206-1209,1984. 21. Steinhelper, M. E., K. L. Cochrane, and L. J. Field. Hypotension in transgenic mice expressing atria1 natriuretic factor fusion genes. Hypertension DuZZus16: 301-307,199O. 22. Steinhelper, M. E., N. A. Lanson, K. P. Dresdner, J. B. and
N.
Delcarpio,
A.
L.
Wit,
W.
C.
Claycomb,
and
L.
J.
Field.
Proliferation in vivo and culture of differentiated adult atria1 cardiomyocytes from transgenic mice. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1826-H1834, 1990.
23
Stewart, T. A., N. B. Hecht, P. G. Hollingshead, P. A. ’ Johnson, J. A. Leong, and S. L. Pitts. Haploid-specific transcription of protamine-myc and protamine-T-antigen fusion genes 24 in transgenic mice. Mol. Cell. Biol. 80: 1748-1755, 1988. l
Swanson, Brinster,
L. W., D. M. Simmons, M. G. Rosenfeld, and
J. Arriza, R. M. Evans.
R. Hammer,
R.
Novel developmental specificity in the nervous system of transgenic animals expressing growth hormone fusion genes. Nature Land. 317: 363366,1985.
25* Takatoh,
H.,
H. Iwamoto, M. and K. Kamoi.
Ikezu,
N. Katoh,
H. Kaneko,
Cardiac rhabdomyoma: a case report with reference to atria1 natriuretic peptide. Actu Puthd. Jpn. 38: 95-104,1988. P.S. Hybridization of denatured RNA and small DNA 26. Thomas, fragments transferred to nitrocellulose. Proc. Nutl. Acud. Sci. USA 77: 5201-5205,198O. G., J. Miller, G. Bencen, and J. Lewicki. Structure 27. Vlasuk, and analysis of the bovine atria1 natriuretic peptide precursor gene. Biochem. Biophys. Res. Commun. 136: 396-403,1986. 28. Wu, J., M. C. LaPointe, B. L. West, and D. G. Gardner. Tissue-specific determinants of human atria1 natriuretic factor H.
Ishikawa,
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING gene expression in cardiac tissue. J. Biol. Chem. 1989. 29. Yamanaka, Brewer, Lewicki,
264:
TUMORS
MICE
E531
cDNA for the rat atria1 natriuretic 308:719-722,1984.
6472-6479,
M., B. Greenberg, L. Johnson, J. Seilhamer, T. Friedemann, J. Miller, S. Atlas, J. Laragh, and J. Fiddes. Cloning and sequence analysis
IN TRANSGENIC
M. J.
of the
30. Zeller,
R., E. Seidman.
K. D. Bloch,
factor precursor. Nature
B. S. Williams,
R. J. Arceci,
Lord. and
C.
Localized expression of the atria1 natriuretic factor gene during cardiac embryogenesis. Genes Deu. 1: 693-698,1987.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
Atria1 natriuretic peptide synthesis in atria1 tumors of transgenie mice. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E524E531,1992.-Transgenic mice harboring a chimeric gene linking mouse protamine 1 W-flanking sequence to the coding sequence of the simian virus 40 T-antigen develop spontaneous rhabdomyosarcomas of the right atria. The presence of the tumors is accompanied by dramatic elevations in plasma atria1 natriuretic peptide (ANP) immunoreactivity (1,698 & 993 vs. 60 & 18 fmol/ml for controls) and hematocrit (56 t 8 vs. 51 t 2 for controls). The immunoreactive ANP (irANP) present in the tumors is similar in size to irANP found in normal mouse atria. ANP mRNA transcripts present in the tumors also appear to be very similar in overall size and 5’-termini to those produced in normal cardiac tissue. Microscopically, the tumors are composed of a disorganized array of densely packed abnormal-appearing cells. Immunocytochemistry and in situ hybridization analysis reveal considerable heterogeneity in ANP gene expression. ANP peptide and mRNA are detectable throughout the parenchyma of the tumors, but absolute levels of expression vary widely among different cells in the population. These tumors represent a potentially valuable model for the study of inappropriate ANP secretion and may provide a tissue source for the development of an ANP-producing atria1 cell line. rhabdomyosarcoma; protamine gene expression; in situ hybridization THEATRIAL NATRIURETIC PEPTIDE (ANP)is ahormone that is synthesized and secreted predominantly by the cardiac atria (1). Its natriuretic and vasorelaxant properties, as well as its well-described antagonism of the renin-angiotensin-aldosterone axis (1, 15), suggest that it represents a naturally occurring antihypertensive peptide that opposes stimuli of volume expansion and increased arterial blood pressure. Recently, it was reported (2) that introduction of a chimeric gene, linking the Y-flanking sequences (5’FS) of a mouse protamine 1 gene (mP1) to the coding sequence of the simian virus 40 (SV-40) T-antigen, into transgenic mice resulted in the seemingly fortuitous development of atria1 rhabdomyosarcomas as well as bilateral osteosarcomas of the petrous portion of the temporal bone. In the current study, we have determined that the capacity for ANP gene expression remains intact in a subpopulation of the transformed atria1 cardiocytes in these tumors and that this expression is accompanied by elevated levels of ANP in the circulating plasma. E524
0193-1849/92
$2.00
Copyright
MATERIALS AND METHODS Materials. Restriction and modification enzymes were obtained from either Bethesda Research Laboratories or Boehringer Mannheim. [a-32P]dCTP (X,000 Ci/mmol) and [cY-~~P]dATP (~400 Ci/mmol) were obtained from Amersham; [T-~~P]ATP (~3,000 Ci/mmol) was purchased from ICN. Antirat (r) ANP antisera were purchased from either Research and Diagnostic (Emeryville, CA) or Peninsula Laboratories (Belmont, CA). Tissue preparation. mPl-SV-40 transgenic mice were originally generated by microinjection of the chimeric gene construct into the pronuclei of C57BL/6 x SJLF2 fertilized eggs. Characterization of these lines has been described (2). The lines were maintained by breeding transgenic animals with C57BL/6 x SJLF, nontransgenic controls. C57BL/6 X SJLF, age-matched mice served as controls for this study, Tumorbearing animals were killed at -10-20 wk of age, and tumors, dissected free of attatched normal myocardium, and ventricular tissue were collected, quick-frozen in liquid nitrogen, and stored at -70°C until processed. Normal atria and ventricles were also collected from nontransgenic mice and pooled for analyses. Hematocrit was measured on freshly collected blood after centrifugation in a bench-top hematocrit centrifuge. For measurement of plasma ANP, trunk blood from freshly killed animals was collected in EDTA, and plasma was obtained by centrifugation at 500 g at 4OC. Preparation and analysis of RNA. Total RNA was extracted from the atria1 tumors or normal cardiac tissue using the method of Cathala et al. (6). RNA was size fractionated on a 1% agarose gel and blot hybridized to an rANP cDNA probe (29) labeled by nick translation [ -lo8 counts. min”(cpm). pg-‘I. Blot hybridizations were carried out as described previously (26), except that prehybridization buffer was used for both the prehybridization and the hybridization steps, the temperature of the hybridization was 32”C, and the wash was in x0.1 standard sodium citrate (0.15 M NaC1/0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate at 23°C. The latter modifications were introduced to optimize the association of the heterologous rANP cDNA probe with the mouse ANP transcript. Autoradiograms were obtained for individual blots, and levels of ANP transcripts were approximated using scanning laser densitometry. Primer extension analysis was carried out as described previously (12) using a 23-nucleotide primer corresponding to residues 545-569 of the complementary strand of the mouse ANP gene (20). This sequence reads 5’-GGGTCGGGGCACGATCTGATGTT-3’. Products were size fractionated on a denaturing 6% polyacrylamide gel. S1 nuclease protection analysis was carried out as described previously (12) using a 5’-end labeled 347-base pair (bp) Hind III-BgZ II fragment, which spans the transcription start site on the mouse ANP gene. The predicted size of the fragment
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING
TUMORS
protecting the &end of the mouse ANP transcript was 194 nucleotides. DNA sequencing. Chain-termination DNA sequencing was carried out on the double-stranded mPl-SV-40 template using a 22nucleotide primer corresponding to positions +80 to +lOl (relative to the transcription start site) in the complementary strand of the SV-40 early-region sequences (11). Sequence was also generated on the alternate strand using a 26-nucleotide primer corresponding to residues +45 to +70 in the mP1 5’FS (18). Each primer provided an identical double-stranded sequence across the mPl-SV-40 junction region. Tissue extract preparation and analysis for ANP immunoreactiuity. ANP was extracted from atria1 tumors and normal atria1 tissue as described by DeBold and Flynn (8). Briefly, frozen tissue was homogenized in 10 vol of an ice-cold extraction solution [ 1 M acetic acid, 1 M HCl, 1% NaCl, pepstatin A (1 mg/l), and phenylmethylsulfonyl fluoride (1 mg/g tissue)] using a polytron PT 35 for 60 s at 50% maximum power. The homogenate was centrifuged at 10,000 g for 30 min. The pellet was reextracted with 5 vol of extraction solution, and the supernatants were combined and frozen at -40°C until highperformance liquid chromatography (HPLC) analysis. Immunoreactive ANP (irANP) in plasma, atria1 tumor, and normal atria1 tissue was measured by a direct radioimmunoassay (RIA) as described (7) using an antiserum to human (h) ANP-(99-126), with the same peptide used as standard (Peninsula Laboratories) and trace (Amersham). HPLC fractions and tissue extracts were assayed directly; plasma samples were assayed after extraction on Cls Sep-Pak cartridges (Waters) as previously described (7). Tissue irANP levels reported in Table 1 were measured on small aliquots of the guanidinium monothiocyanate (GSCN) suspension used for isolation of RNA (see above). GSCN had no effect on the RIA at concentrations present in the assay mixture. Samples were resuspended in 100 rug assay buffer (0.1 M NaCl, 0.05 M sodium phosphate, pH 7.2, 0.1% bovine serum albumin, 0.01% Triton X-100, 0.01% NaN& and were assayed using a rabbit anti-hANP antiserum (Research and Diagnostics). The latter antibody displayed 30% cross-reactivity with human pro-ANP; the Peninsula antibody was 40% cross-reactive with the prohormone. Both antibodies employed have 100% cross-reactivity to rANP; the latter is identical in sequence to mouse ANP (20,27). Extracts of plasma, atria1 tumor, and normal mouse atria were analyzed by reverse-phase HPLC using a Cls PBondapak Table 1. Immunoreactiue of transgenic mice Sample
ANP levels in tumors irANP, pg ANP/pg soluble protein
Pooled normal atria 15.18 Pooled normal ventricles 0.25 B75 Tumor 7.01 Ventricle 0.36 B77 Tumor 15.57 Ventricle 0.36 B79 Tumor 3.05 Ventricle 0.21 B80 Tumor 15.33 Soluble protein was prepared from homogenized tissue (i.e., tumor or normal ventricular tissue from same animal) as described in MATERIALS AND METHODS and was assayed for immunoreactive atria1 natriuretic peptide (irANP) using a Research and Diagnostic anti-rat ANP antisera. See legend to Fig. 1 for identification of individual transgenic animals (B75, B77, B79, and B80).
IN TRANSGENIC
MICE
E525
column (Waters) developed at a flow rate of 1 ml/min with a gradient of lo-60% acetonitrile in 0.1% trifluoroacetic acid over 50 min. One-minute fractions were collected; eluates were dried in a Speed Vat evaporator and reconstituted in buffer immediately before RIA. After each injection, the retention time of 1251-labeled hANP was determined as a reference marker. Differences were documented by analysis of variance and the Newman-Keuls test for significance or by Student’s t test. Immunocytochemistry and in situ hybridization histochemistry. Excised hearts bearing the atria1 tumors were placed directly into Bouin’s sublimate, fixed, sectioned, and processed as described previously (12). Immunostaining was carried out using a rabbit anti-rANP antiserum (AP l-2) raised against thyroglobulin-coupled rANP. The titer of the antibody was 1:60,000 as determined by binding of 12’1-labeledrANP to serial dilutions of the antiserum. The antiserum was used at a l:l,OOO dilution for the immunocytochemical studies. As a negative control, the antiserum, at a l:l,OOO dilution, was preabsorbed to the rANP peptide (final concentration 10 pg/ml) before incubation with the atria1 section. This resulted in the elimination of immunostaining in the section (data not shown). For the hybridization studies, atria1 tumors, normal atria, and ventricles were embedded in OCT compound and frozen immediately in a methanol-dry ice bath. Subsequent preparation and hybridization to a [3H]cRNA probe for rANP were carried out as described previously (9). Sections were exposed for 3-4 wk before development of the photographic emulsion. Hybridization with a [3H]mRNA rANP probe revealed only background levels of hybridization in each of the tissues examined (data not shown). RESULTS
Plasma ANP levels in five transgenic mice bearing atria1 tumors averaged 1,698 t 993 (SD) fmol/ml. These levels were -30-fold higher than levels in control mice (60 t 18 fmol/ml; P < 0.01 ) of the same strain maintained on the same diet. Measurements of hematocrit, as an assessment of ANP bioactivity in vivo (1, 15), were made on transgene-bearing mice as well as a similar number of their nontransgenic litter mates. Hematocrits were, in general, elevated in the transgenic group [56 t (SD) 8 vs. 51 t 2 in the nontransgenic animals; P < 0.01; n = 26 for the former and 25 for the latter group], raising the possibility that the elevations in ANP promote significant alterations in volume homeostasis. Given the large size of these tumors, elevations of plasma ANP in these mice could arise either directly through secretion from the atria1 tumor cells or from tumor-related obstruction to atria1 outflow with elevated intra-atria1 pressure and increased ANP release from the neighboring atria1 tissue. To address this issue, we examined the tumor tissue for the presence of the ANP peptide as well as the ANP mRNA transcript. As shown in Fig. 1, the atria1 tumors possess significant quantities of ANP mRNA, which is of the same size and 40-15% the abundance of that produced in the normal cardiac atria. The cardiac ventricles, on the other hand, contained only small amounts of the ANP mRNA, a finding similar to that reported earlier for rats (12). The high levels of ANP mRNA were accompanied by high levels of irANP protein in the tumors (Table 1). Extracted plasmas from the transgenic and control animals were subjected to HPLC analysis. As shown in
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
E526
ANP-PRODUCING
TUMORS
IN
TRANSGENIC
MICE
123456789
Fig. 1. Blot-hybridization analysis of atria1 natriuretic peptide (ANP) transcripts in RNA from atria1 tumors. Total RNA from pooled normal mouse atria (3 fig, lane l), pooled normal mouse ventricle (30 pg, Lane 2), ventricle from mouse B75 (20 pg, lane 3), atria1 tumor from B75 (20 pg, lane 4), ventricle from B77 (20 pg, lane 5), atria1 tumor from B77 (20 pg, lane 6) ventricle from B79 (20 pg, lane 7), atria1 tumor from B79 (20 Fg, lane 8), or atria1 tumor from B80 (20 wg, lane 9) was size fractionated, blotted to nitrocellulose filters, and hybridized to rat ANP cDNA probe. Transcripts were -950 nucleotides in size based on comparison with DNA markers run in parallel. Subsequent reprobe of blot with radiolabeled actin cDNA revealed roughly equivalent levels of actin transcript in each tumor RNA lane. Animal B75, 18-wk-old male from 1738-3 line; B77, 16-wk-old male from 1736-l line; B79, 16-wk-old male from 1738-4 line; BSO, 15-wk-old male from 1736-8 line.
Fig. 2 the major form(s) of irANP in the plasma of the normal (Fig. 20) and transgenic mice (Fig. 2B) migrated very close to the position of hANP-(99-126), eluting l2 min ahead of the ‘251-labeled standard. On the other hand, extracts from either normal mouse atria (Fig. 2C) or the mouse atria1 tumors (Fig. 2A) contained large amounts of late-eluting immunoreactivity, corresponding to the ANP precursor [ANP-(l-126); retention time 3538 min] in addition to mature ANP and a variety of intermediate forms (5). Broadening of the initial portion of the immunoreactive peaks presumably represents
4
lz51hANP 4 P I/ I/
NH2-terminally degraded metabolites that are detected by the antibody. To assure that the observed expression was being driven from the native ANP gene promoter, we carried out primer extension and nuclease Si protection analyses on RNA from the atria1 tumors. As shown in Fig. 3A, primer extension produced products 82-86 nucleotides in length. This corresponded to 5’-termini 23-27 bp downstream from the TATA box sequence in the mouse ANP gene (20), which is believed to dictate the initiation of the dominant ANP transcript. This start was also rf;000
4000 Cl 2000
60
-I
18
Retention
time
6 2 3 =I
Fig. 2. Reverse-phase high-performance liquid chromatography of extracts of plasma and atria1 tissue from tumorbearing transgenic mice and normal control mice. Plasma or tissue extracts injected were equivalent to 12 mg atria1 tumor (A), 30 ~1 transgenic mouse plasma (B), 215 fig normal mouse atria (C), and 190 ~1 normal mouse plasma (D). Broken lines, elution pattern of iZ51labeled human ANP (hANP; 10,000 counts/min) injected after each experiment (scale shown at right). Major early eluting peaks of immunoreactive ANP (irANP) had retention time (18-20 min) consistent with that of ANP-(99-126); as expected, elution of ‘251-labeled standard was delayed by l-2 min.
(mln)
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING
12
3
4
5
TUMORS
IN
TRANSGENIC
MICE
6
-242 >
-190
E527
Fig. 3. Identification of 5’-termini of mouse ANP transcripts in atria1 tumor. A: primer extension analysis of total RNA from normal mouse ventricle (20 pg, lane 1 ), normal atria (1 rg, lane 2) or atria1 tumor from B75 (20 pg, lane 3), B77 (20 pg, lane 4), B79 (20 pg, lane 5), and B80 (20 rg, lane 6). Arrow, position of dominant mouse ANP transcript. B: nuclease S, protection of total RNA from normal atria (3 pg, lane I), normal ventricle (20 pg, lane 2) or atria1 tumors from B75 (30 pg, lane 3), B77 (30 wg, lane 4), and B80 (30 gg lane 5). Numbers to right represent size in base pairs of DNA markers run in parallel.
-147
identified in RNA from normal atria1 and ventricular tissue (Fig. 3A). Nuclease Si protection was carried out using a 347-bp Hind III-Bgl II fragment that spans the 5’-end of the mouse ANP gene. This probe (Fig. 3B) protected a fragment -195 nucleotides in length, with RNA from atrial, ventricular, or tumor tissue, mapping the start site to a position overlying that demonstrated by primer extension above. Finally, to confirm that expression of the ANP gene was carried out within the tumor cells, we employed immunocytochemistry and in situ hybridization histochemistry to localize ANP gene expression at a cellular level. Immunocytochemical analysis for irANP (Fig. 4A) in the normal atria revealed a fairly homogenous pattern of immunostaining with a dense perinuclear concentration of the peptide. In the case of the tumor section, the staining pattern was considerably more heterogenous (Fig. 4C). Individual cells scattered throughout the tumor parenchyma stained to varying degrees for ANP immunoreactivity. Positive cells tended to be clustered together in a swirling pattern coursing among cells displaying much lower, or nonexistent levels of irANP. Hybridization of normal mouse atria1 sections with an rANP [3H]cRNA probe revealed a relatively intense signal that spread across the full thickness of the atria1 wall (Fig. 4B), mirroring the distribution of immunoreactivity. A similar pattern of expression has been reported previously in the l-day-old mouse heart (30). The signal was very low to absent in the mouse ventricle (data not shown). Hybridization of the [3H]cRNA to sections of the atria1 tumor (Fig. 40) revealed a much more heterogenous pattern with areas of high-intensity signal superimposed on a lower-intensity background. This signal was in general lower than that seen in “normal” atria1 tissue (see Fig. 4C) but considerably higher than that found in the cardiac ventricle (data not shown). The regions displaying the most intense hybridization activity followed a swirling pattern that resembled that described above for ANP immunoreactivity. DISCUSSION
As mentioned above, the expression of the mPl-SV40 transgene results in the seemingly fortuitous appear-
ance of atria1 and temporal bone tumors (2). The expression of T-antigen was more pronounced in the atria1 tumors than it was in round spermatids of the testes, cells that usually employ the mP1 promoter with a high degree of efficiency (18). It is noteworthy that testicular tumors were absent. Previous investigations have provided no evidence suggesting increased expression of either the endogenous mP1 gene (2, 14) or an mP1 transgene (18) in cardiac tissues. In addition, numerous studies employing a variety of SV-40 T-antigen-containing transgenes have failed to provide evidence for selective expression of these sequences in cardiac tissue (17). Thus it appears that expression of the mPl-SV-40 gene results from the utilization of a tissue-specific element(s) that may have been created fortuitously by linking the SV-40 and protamine gene sequences. Such novel expression patterns have been noted previously in transgenic mice harboring chimeric metallothionein-growth hormone fusion genes (19, 24). This could result either from the synergistic interaction of normally quiescent sequences present in each of these genes or alternatively from the de novo generation of a unique element at the point of fusion of these two genes. To examine the latter possibility, DNA sequence analysis was carried out across the mPl-SV-40 junction region. This sequence is presented in Fig. 5. We have compared the sequence of the junction region with that of a DNA segment from the hANP gene (i.e., positions -385 to -339), which we have recently shown by a combination of functional and gel mobility-shift analyses to be important in regulating expression of that gene (28). As shown in Fig. 5, a region of shared homology can be identified. When aligned for maximal overlap, the two sequences are homologous at 19 of 28 positions. In the case of the mPl-SV-40 construction, the homologous sequence is derived entirely from the mP1 gene and polylinker-adapter sequence. These findings raise the possibility that linkage of the protamine and SV-40 gene sequences through a synthetic adapter-linker may have contributed, in part, to the creation of a cis-acting regulatory element that, in the context of the surrounding transgene sequence, allows for increased expression of the viral T-antigen and consequent expression of the transformed phenotype in
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
E528
ANP-PRODUCING
TUMORS
IN
TRANSGENIC
MICE
Fig. 4. A: immunocytochemistry of ANP on section of normal mouse atrium. No hematoxylin counterstaining. Immunostaining is spread homogeneously across section. Particularly dense concentrations of staining are noted in a perinuclear distribution. Magnification ~63. Bar = 75 pm. B: in situ hybridization histochemistry of section of normal mouse atrium, using rat ANP [3H]cRNA probe; photographed in darkfield illumination. Cell nuclei were counterstained with hematoxylin. Note that a small rim of connective tissue with more densely packed nuclei can be seen on right corner (see arrow) where no signal is present. No signal was observed when a rat ANP [3H]mRNA probe was used on adjacent sections (not shown). Magnification x40. Bar = 100 pm. C: immunocytochemistry of ANP on transgenic mouse atria1 tumor section. No hematoxylin counterstaining. Positive immunoreactivity appears as dark deposits of diaminobenzidine (see arrows for example of immunopositive cells). Appearance of tumor section is that of swirls of spindle-shaped cells separating clusters of more densely packed cells. Although ANP immunoreactivity was detected throughout tumor, it was especially dense in these cellular swirls where ratio of cytoplasmic to nuclear area was highest. Magnification x40. Bar = 100 pm. D: in situ hybridization histochemistry on transgenic mouse atria1 tumor section using rANP [3H]cRNA probe. Cell nuclei were counterstained with hematoxylin. Distribution of signal in atria1 tumor was similar to what was observed with immunocytochemistry [i.e., while scattered throughout tumor, signal was more intense in those areas harboring swirls of spindle-shaped cells (see arrows)].
atria1 cells. However, the mechanism is likely to be more complicated than this. Field (10) recently reported that introduction of a chimeric construction linking 500 bp of 5’FS from the hANP gene, a segment that includes the tissue-specific element (28) alluded to above, to the SV-40 T-antigen coding sequences resulted in the generation of grossly enlarged right atria. Pathologically these atria were characterized by hyperplasia rather than the rhabdomyosarcomas seen with the mPl-SV-40 transgene. In addition, Stewart et al. (23) recently reported the generation of transgenic mice bearing the regulatory sequences of the mP2 gene, which bears a high degree of sequence homology to mP1 (13), linked to the coding sequences of the SV-40 T-antigen. In that case the transgene was expressed in cardiac cells, but tumorigenesis was not reported (23). Thus there are in all likelihood additional factors present in the mPl-SV-40 construct
that are responsible for the complete manifestation of the malignant phenotype. Of additional interest is the finding that both the hANF-SV-40 Tag gene of Field (10) and the mPl-SV-40 Tag gene described by Behringer et al. (2) appear to express the neoplastic phenotype preferentially in the right vs. left atria. This could imply selective chamber-specific utilization of transcriptional regulatory signals in the chimeric genes. Because the endogenous ANP gene shows no such preferential expression, these signals are likely to involve elements outside the ANP promoter or the homologous element identified in the mPl-SV-40 Tag fusion gene (see above). An alternate possibility is that expression of these genes alone may be necessary but not sufficient for malignant transformation. Exposure to a second growth-promoting factor, with a topographically limited distribution of expression, might provide the requisite stimulus for dem-
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING
TUMORS
IN TRANSGENIC
E529
MICE -337
+81
+123 n I
mpl
n n
SFS
Adapter/Bgl II sv40 Linker Fig. 5. Sequence of mP1-SV-40 fusion region. Sequence from sense strands of hANP 5’FS and fusion region of mPlSW40 construction are presented. hANP sequence is numbered relative to major transcription start site in hANP gene. mP1 sequence is numbered relative to mP1 start site, which appears to be employed in chimeric gene (3). Contributions of mP1 YFS, synthetic linker-adapter, and SV-40 T-antigen coding sequences are delineated below mPl-W-40 sequence. Sequences have been aligned for maximal overlap.- Regions with 2 or more sequential homologous bases are shaded.
onstration of the full neoplastic phenotype. The latter hypothesis is supported by the studies of Field (lo), alluded to above, in that he found evidence for Tag expression in both the right and left atria despite the strong predilection for neoplasia on the right side. The extreme elevations in plasma ANP noted in several of the mice were sufficient to lead to a gross disturbance in volume regulation as reflected by the elevations in hematocrit. Steinhelper et al. (21) recently reported on the generation of a mouse line harboring a transgene linking the mouse transthyretin promoter to the structural gene for ANP. These mice produced large amounts of pro-ANP in the liver. The latter finding was expected since the liver avidly expresses th ,e endogenous transthyretin gene. PI .asma ANP leve 1s in these animals were elevated significantly, and, most importantly, they displayed a number of physiological characteristics suggestive of chronic ANP excess. These findings, similar to those described in the present study, suggest that chronic endogenous hypersecretion of ANP can result in sustained physiological perturbations in cardiovascular homeostasis. Although we cannot rigorously exclude a contribution from juxtapositioned normal or near-normal atria1 tissue to the elevation in circulating plasma ANP levels, the presence of ANP transcripts and ANP immunoreactivity within the tumor mass strongly suggests that the elevated plasma levels result, at least in part, from synthesis of ANP within the tumor itself. In addition, preliminary studies indicate that cultured explants of tumor tissue continue to secrete irANP for several months after removal from the host animals (S. Hauschka and C. Gartside, personal communication), supporting the hypothesis that the elevated plasma levels derive from tumor production of ANP. ANP mRNA levels in most of the tumors were somewhat less than those found in normal atria1 tissue (Fig. 1) but considerably higher than that found in either pooled ventricular tissue from normal animals or ventricular tissue taken from the same transgenie mice. Given the large size of these tumors relative to the normal atria, it is likely that tumor-directed ANP synthesis represents a significant fraction of total ANP production in these mice. One exception was the tumor RNA sample shown in lane 8 of Fig. 1, where ANP transcripts were not seen. ANP mRNA was detectable in the more sensitive primer extension analysis (Fig. 3A, lane 5) but was still considerably less than that found in
the other tumor RNA preparations. This does not reflect degradation of the RNA or methodological problems with the analysis, since rehybridization of the blot to a radiolabeled human actin cDNA probe demonstrated roughly equivalent levels of actin transcript in each of the tumor RNA preparations (data not shown). This same tumor demonstrated the lowest level of ANP immunoreactivity in the group, although it was still considerably higher than ventricular levels. These findings suggest that the heterogeneity seen in plasma ANP levels may result not only from differences in individual tumor mass within the transgenic population but also from intrinsic variability among the tumors in terms of their capacity to express the ANP gene and synthesize the encoded protein. Variability in the latter case may reflect cellular heterogeneity in the levels of mANP gene expression which, based on the immunocytochemical and in situ hybridization studies, appears to vary considerably among the different cells in the population. The reasons underlying this heterogeneity given a common genotypic background among the cells remains unknown. The overall size and 5’-termini of the tumor transcripts appear to be similar if not identical to those of the normal mouse atria, indicating that expression of the ANP gene is being driven from its native promoter. In addition, extracts of tumor, like normal atria, contained large amounts of pro-ANP and other high-molecularweight forms, whereas the mature hormone predominated in plasma (Fig. 2). These findings imply that introduction of the transgene has not resulted in gross alterations in ANP gene expression per se but rather that the transformation has resulted in an overproliferation of ANP-producing atria1 cells. A fraction of these transformed atriocytes, at least at the time of tumor resection, appear to have retained the ability to express the ANP gene at a modestly reduced level and to process the prohormone product appropriately. Interestingly, a recent case report of a patient with multicentric rhabdomyoma of the heart (25) described the presence of ANP immunoreactivity in the atria1 but not ventricular lesions, a finding that supports the hypothesis put forth above (i.e., retention of the atria1 phenotype in the neoplastic cells). The implications of the availability of these tumors are numerous. Selective generation of tumors through the transgene approach has been shown to provide a useful source of transformed cell lines retaining charac-
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
E530
ANP-PRODUCING
TUMORS
teristics of the tissue of origin (16). This has particular importance in terms of the atria1 myocytes since no continuous culture line presently exists. Employing transplanted hyperplastic atria1 tissue fragments from their hANF-SV-40 Tag-bearing transgenic mice as a tissue source, Steinhelper et al. (22) succeeded in establishing atria1 myocyte cultures capable of maintaining a differentiated phenotype and replicating in vitro; however, these cells remain difficult to passage continuously in culture. Thus a transformed ANP-producing atria1 rhabdomyosarcoma cell line may offer an additional advantage in terms of facilitating studies of ANP secretion and/or biosynthesis in vitro. In addition, these tumors may represent a viable model of inappropriate ANP hypersecretion, with the potential for providing valuable information regarding the long-term effects of high circulating levels of ANP on volume status and fluid and electrolyte homeostasis in a tumor-bearing animal. Such information could take on clinical importance, given the recent demonstration of ectopic ANP production in a number of human lung carcinomas (3). We thank Bill Pugh for preparation of the manuscript. We also thank J. Wu for assistance with the sequence analysis and B. Hedges, K. Nguyen, and K. Nakamura for technical assistance. We are also grateful to J. Peschon for providing us with the chimeric plasmid (mPlSV-40) and SV-40 oligonucleotide primer employed in DNA sequencing studies, to L. Field for providing the mouse ANP probe used in the nuclease S1 protection study, and to L. Kedes for providing the human actin cDNA. This work was supported by National Heart, Lung, and Blood Institute Grants HL-35753, HL-38774, and HL-18323SCR and by a Grant-in-Aid from the American Heart Association, with partial support from the Redwood Empire Chapter. D. G. Gardner is an Established Investigator for the American Heart Association. Address for reprint requests: D. G. Gardner, Dept. of Medicine, Metabolic Research Unit, Univ. of California, San Francisco, CA 94143. Received 3 September 1991; accepted in final form 13 November 1991. REFERENCES 1. Baxter, J, D., J. A. Lewicki, and D. G. Gardner. Atria1 natriuretic peptide. Biotechnology 6: 529-546, 1988. 2. Behringer, R. R., J. J. Peschon, A. Messing, C. L. Gartside, S. D. Hauschka, R. D. Palmiter, and R. L. Brinster. Heart and bone tumors in transgenic mice. Proc. A&l. Acud. Sci. USA 85: 2648-2652,1988. R. I. Linnoila, M. J. Birrer, A. F. 3. Bliss, D. P., J. F. Battey, Gazdar, and B. E. Johnson. Expression of the atria1 natriuretic factor gene in small cell lung cancer tumors and tumor cell lines. J. Nutl. Cancer Inst. 82: 305-310,199O. R. L., H. Y. Chen, M. E. Trumbauer, M. K. Yagle, 4, Brinster, and R. D. Palmiter. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Nutl. Acud. Sci. USA 82: 4438-4442,1985. M. J. F., J. H. Laragh, and S. A. Atlas. Character5. Camargo, ization of atria1 natriuretic factor released by cultured human atria1 myocytes, In: Biologically Active Atria1 Peptides, edited by B. M. Brenner and J. H. Laragh. New York: Raven, 1987, vol. I, p. 2l26. G., J. F. Savouret, B. L. Mendez, M. Karin, J. A. 6. Cathala, Martial, and J. D. Baxter. A method for isolation of intact translationally active ribonucleic acid. DNA NY 2: 329-335, 1983. Cody, R. J., S. A. Atlas, J. H. Laragh, Covit, K. S. Ryman, A. Shaknovitch, F. Camargo, R. M. Scarborough, and
S. H. Kubo, K. Pandolfino, J. A. Lewicki.
A. B. M. J.
Atria1 natriuretic factor in normal subjects and heart failure patients. J. Clin. Invest. 78: 1362-1374, 1986. DeBold, A. J., and T. G. Flynn. Cardionatrin I-a novel peptide
IN TRANSGENIC
MICE
with potent diuretic and natriuretic 302,1983. 9. Deschepper,
properties. Life Sci. 33: 297-
C. F., S. J. Mellon, J. Jen, and Y. F. Lau.
T. L.
Reudelhuber,
D. G.
In situ hybridization histochemistry on mRNA in endocrine tissues. In: Selected Topics in lMoZecuZurEndocrinology,. edited by Y. F. Lau. New York: Oxford Univ. Press, 1987, p. 31-41. T antigen transgenes 10. Field, L. J. Atria1 natriuretic factor-SV40 produce tumors and cardiac arrhythmias in mice. Science Wash. DC 239: 1029-1033,1988. 11. Fiers, W., R. Contreras, G. Haegeman, R. Rogiers, A. Van Gardner,
de Voorde, G. Volckaert,
H. Van Heuverswyn, and M. Ysebaert.
H. Swyn,
J. Herreweghe,
Complete nucleotide sequence of SV40 DNA. Nature Land. 273: 113-120,1978. 12. Gardner,
D. G., C. F. Deschepper, W. F. Ganong, S. Hane, J. D. Baxter, and J. Lewicki. Extra-atrial expres-
J. Fiddes,
sion of the gene for atria1 natriuretic USA 84: 2175-2179,1986. 13. Johnson,
P. A., J. J. Peschon, B. Hecht. Sequence
factor. Proc. Nutl. Acud. Sci.
P. C. Yelick,
R. D. Palmiter,
homologies in the mouse protamine 1 and 2 genes. Biochim. Biophys. Actu 950: 45-53,1988. 14. Kleene, K. C., R. J. Distel, and N. B. Hecht. cDNA clones encoding cytoplasmic poly(A)+ RNAs which first appear at detectable levels in haploid phase of spermatogenesis in the mouse. Deu. Biol. 98: 455-464, 1983. 15. Maack, T., M. J. F. Camargo, H. D. Kleinert, J. H. Laragh, and S. A. Atlas. Atria1 natriuretic factor: structural and functional properties. Kidney Int. 27: 607-615, 1985. 16. Ornitz, D. M., R. E. Hammer, A. Messing, R. D. Palmiter, and R. L. Brinster. Pancreatic neoplasia induced by SV40 T antigen expression in acinar cells of transgenic mice. Science Wash. DC 238: 188-193,1987. 17. Palmiter, R. D., H. Y. Chen, A. Messing, and R. L. Brinster. SV40 enhancer and large-T antigen are instrumental in development of choroid plexus tumors in transgenic mice. Nature Land. 316: 457-460,1985. 18. Peschon, J. J., R. R. Behringer, R. L. Brinster, and R. D. Palmiter. Spermatid-specific expression of protamine 1 in transgenie mice. Proc. Nutl. Ad. Sci. USA 84: 5316-5319,1987. 19. Russo, A. F., E. B. Crenshaw, S. A. Lira, D. M. Simmons, L. W. Swanson, and M. G. Rosenfeld. Neuronal expression of chimeric genes in transgenic mice. Neuron 1: 311-320, 1988. 20. Seidman, C., K. Bloch, K. Klein, J. Smith, and J. Seidman. Nucleotide sequences of the human and mouse atria1 natriuretic factor genes. Science Wash. DC 226: 1206-1209,1984. 21. Steinhelper, M. E., K. L. Cochrane, and L. J. Field. Hypotension in transgenic mice expressing atria1 natriuretic factor fusion genes. Hypertension DuZZus16: 301-307,199O. 22. Steinhelper, M. E., N. A. Lanson, K. P. Dresdner, J. B. and
N.
Delcarpio,
A.
L.
Wit,
W.
C.
Claycomb,
and
L.
J.
Field.
Proliferation in vivo and culture of differentiated adult atria1 cardiomyocytes from transgenic mice. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1826-H1834, 1990.
23
Stewart, T. A., N. B. Hecht, P. G. Hollingshead, P. A. ’ Johnson, J. A. Leong, and S. L. Pitts. Haploid-specific transcription of protamine-myc and protamine-T-antigen fusion genes 24 in transgenic mice. Mol. Cell. Biol. 80: 1748-1755, 1988. l
Swanson, Brinster,
L. W., D. M. Simmons, M. G. Rosenfeld, and
J. Arriza, R. M. Evans.
R. Hammer,
R.
Novel developmental specificity in the nervous system of transgenic animals expressing growth hormone fusion genes. Nature Land. 317: 363366,1985.
25* Takatoh,
H.,
H. Iwamoto, M. and K. Kamoi.
Ikezu,
N. Katoh,
H. Kaneko,
Cardiac rhabdomyoma: a case report with reference to atria1 natriuretic peptide. Actu Puthd. Jpn. 38: 95-104,1988. P.S. Hybridization of denatured RNA and small DNA 26. Thomas, fragments transferred to nitrocellulose. Proc. Nutl. Acud. Sci. USA 77: 5201-5205,198O. G., J. Miller, G. Bencen, and J. Lewicki. Structure 27. Vlasuk, and analysis of the bovine atria1 natriuretic peptide precursor gene. Biochem. Biophys. Res. Commun. 136: 396-403,1986. 28. Wu, J., M. C. LaPointe, B. L. West, and D. G. Gardner. Tissue-specific determinants of human atria1 natriuretic factor H.
Ishikawa,
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
ANP-PRODUCING gene expression in cardiac tissue. J. Biol. Chem. 1989. 29. Yamanaka, Brewer, Lewicki,
264:
TUMORS
MICE
E531
cDNA for the rat atria1 natriuretic 308:719-722,1984.
6472-6479,
M., B. Greenberg, L. Johnson, J. Seilhamer, T. Friedemann, J. Miller, S. Atlas, J. Laragh, and J. Fiddes. Cloning and sequence analysis
IN TRANSGENIC
M. J.
of the
30. Zeller,
R., E. Seidman.
K. D. Bloch,
factor precursor. Nature
B. S. Williams,
R. J. Arceci,
Lord. and
C.
Localized expression of the atria1 natriuretic factor gene during cardiac embryogenesis. Genes Deu. 1: 693-698,1987.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on January 19, 2019.
