Atrial Natriuretic Factor and Arginine Vasopressin Production in T\Imor Cell Lines from Patients with Lung Cancer and Their Relationship to Serum Sodium1

Home , Natriuretic peptide, Vasopressin

ICANCHR RKSEARCH 53. 67-74. January 1. 1993] Atrial Natriuretic Factor and Arginine Vasopressin Production in T\imor Cell Lines from Patients with Lung Cancer and Their Relationship to Serum Sodium1

Andrew J. Gross, Seth M. Steinberg, J. GarreÂ« Reilly, David P. Bliss, Jr., John Brennan, Phong Tram Le, Alfreda Simmons, Ruby Phelps, James L. Mulshine, Daniel C. Ihde, and Bruce E. Johnson2

National Cancer Institute-Navy Medical Oncology Branch timi Department of Medicine. Uniformed Sen'ices University' of the Health Sciences, and the National Naval Medical Center. BethesJii. Maryland 20889-5105 Â¡A.J. C.. D. P. B.. J. B.. P. T. L, A. S.. K. P., J. L M., D. C. I., B. E. ].]: Biostatistics and Data Management Section. National Cancer Institute. Bethesda. Maryland 20892 Â¡S.M. S./: and Department of Medicine. Washington Hospital Center. Washington. DC 20422 Â¡J.C. R.]

ABSTRACT (6), tumor cell lines (3), or plasma (19, 20). Recent evidence suggests that the ectopie production of ANF may also be involved in SIADH. Patients with lung cancer (n = 263) were studied to determine the Atrial natriuretic factor was isolated from the right atrium in 1981 relationship among ectopie production of atrial natriuretic factors (ANF) (27), and the gene and peptide were subsequently sequenced in the rat and arginine vasopressin (AVP), serum sodium, and patient outcome. Of (28-30) and human (31, 32). Ectopie production of ANF mRNA in 133,21 (16% Â»patientswith small cell lung cancer (SCLC) had hyponatremia (serum sodium, <130 mmol/liter), compared to none of 130 (0%) tumors and tumor cell lines from patients with SCLC and hyponatre patients with non-small cell lung cancer hyponatremia had shorter survival than patients (3). The ANF peptide has been detected in SCLC tumors (33) and with extensive stage SCLC and normal serum sodium values (/' = 0.012). tumor cell lines (3) by radioimmunoassay. Characterization of the Of the 11 h\ ponili mnk patients with SCLC and tumor cell lines available peptide from an SCLC tumor by gel chromatography showed the for study, 9 produced ANF mRNA, 7 of 11 produced AVP mRNA, and 5 of peptide to be similar to the bioactive 28-amino acid form present in 11 produced both ANF mRNA and AVP mRNA. All 11 cell lines produced the plasma (33). Plasma ANF levels have been found to be elevated in either ANF mRNA and ANF peptide or AVP mRNA and AVP peptide, or patients with lung carcinoma and hyponatremia with elevated plasma both. The quantity of AVP peptide in the tumor cell lines was more closely associated with hyponatremia in the patients i/' = 0.0026, /-' = 0.28) than AVP levels (16, 22, 23, 33, 34) and in a single patient with normal was the production of ANF peptide (/' = 0.066, r' = 0.12), although neither plasma AVP levels (35). association was strong. All tumor cell lines studied from SCLC patients While ectopie production of ANF has been documented in tumors with hyponatremia produce ANF and/or AVP mRNA and peptides. and tumor cell lines from patients with small cell lung carcinoma and SIADH, the potential contribution of ANF to hyponatremia has not INTRODUCTION been extensively studied in a prospectively defined cohort of patients with lung cancer. Previous studies of SIADH in a series of patients Abnormalities of serum sodium levels commonly occur in patients have only studied the presence of ANF or AVP in the plasma or urine with SCLC,' most typically recognized as the SIADH (1). Studies of patients by radioimmunoassay without studying ectopie hormone have indicated that ectopie production of AVP by small cell lung production of ANF and AVP by the cancer cells (13-16, 18-21, 34). cancer giving rise to elevated plasma levels of AVP can cause SIADH. The consistent efforts to establish tumor cell lines from our lung Production of AVP mRNA in tumors and tumor cell lines from patients cancer patients treated on protocol studies allows us to study ANF and with SCLC and hyponatremia has been documented by Northern blot AVP mRNA expression and peptide hormone production in tumor cell and S|-nuclease analysis (2, 3). The AVP peptide has been detected in lines from a high proportion of our patients. In addition, we had SCLC tumors and tumor cell lines by bioassay of antidiuretic activity obtained pretreatment serum and urine chemistry values, including (4-8) and by radioimmunoassay (3, 6, 9-11). Biosynthesis of AVP by sodium, and accurate follow-up information concerning these pa SCLC tumor extracts (10) and secretion of AVP by SCLC tumor cell tients. Patients with SCLC and hyponatremia live a shorter period of lines into media (12) has also been demonstrated. Elevated levels of time than patients with SCLC and normal serum sodium values AVP in the urine (13-16) and plasma (16-23) of patients with small (36-38), and hyponatremia is more common in patients with exten cell lung carcinoma and SIADH have been shown in multiple series. sive-stage than limited-stage disease. Therefore, we compared the Infusion of AVP or pitressin with water ingestion causes hyponatremia incidence in our limited and extensive stage patients to the survival of and hypoosmolality in the plasma, as well as hyperosmolality in the the SCLC patients with and without hyponatremia. urine, in humans (24), dogs (25), and rats (26). Therefore, ectopie Some patients with SCLC and hyponatremia often do not fulfill all production of AVP in patients with small cell lung carcinoma has been the criteria of SIADH, and hormones other than AVP may cause shown to play a causal role in hyponatremia. abnormally low serum sodium values. Therefore, we decided to clas Small cell lung cancer patients with hyponatremia have been iden sify our patients as being hyponatremic (serum sodium, <130 mmol/ tified who have no detectable immunoreactive AVP in their tumors liter) or normonatremic. Hyponatremia is a more easily defined con dition which commonly reflects the presence of SIADH and may Received 7/8/92; accepted 10/14/92. potentially be associated with other hormones responsible for the The costs of publication of this article were defrayed in part by the payment of page disruption of sodium homeostasis and hyponatremia. Therefore, we charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this faci. conducted a study of ANF and AVP mRNA expression in all available ' This is a U.S. government work. There are no restrictions on its use. The opinions and cell lines established from patients with SCLC and hyponatremia and assertions contained herein are the private views of the authors and are not construed as compared these data of expression to mRNA expression in cell lines official or as reflecting the views of the Department of the Navy or the Department of Defense. established from patients with SCLC and normal serum sodium val 2 To whom requests for reprints should be addressed, at National Cancer Institute-Navy ues. Arginine vasopressin can stimulate the growth of small cell lung Medical Oncology Branch. Nalional Naval Medical Center. Bldg 8, Rm 5105. Bethesda, cancer in vitro (39-40) which may explain the association between MD 20889-5101. *The abbreviations used are: SCLC. small cell lung cancer; ANF, atrial natriuretic hyponatremia and shorter survival. Therefore, we studied the survival factor; AVP. arginine vasopressin; NSCLC. non-small cell lung cancer; SIADH. syndrome of patients with hyponatremia and AVP production compared to pa of inappropriate antidiuretic hormone; RIA. radioimmunoassay; ACTH. adrenocortico- tropic hormone; BUN. blood urea nitrogen; PBS. phosphate-buffered saline; BNP, brain tients with ANF production. We also studied cell lines from patients natriuretic peplide. with non-small cell lung cancer and normal serum sodium values, 67

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1993 American Association for Cancer Research. ANF AND AVP PRODUCTION IN PATIENTS WITH I.UNG CANCER because of the infrequent occurrence of SIADH in NSCLC patients were cemrifuged at 21.000 x g at 5Â°Cfor 30 min. The supernatant was saved, (1). Radioimmunoassay data from tumor cell line lysates were ob and 3 ml cold PBS was added to the pellet, rehomogenized, and centrifuged again at 21.OCX)xg at 5Â°Cfor 30 min. The supernatant was added to that saved tained in order to confirm the presence of immunoreactive ANF and AVP in tumor cell lines. previously. The soluble protein content of the supernatant was determined by colorimetrie protein assay according to the instructions of the manufacturer (Bio-Rad. Richmond. CA). The 8 ml was applied to individual Sep-Pak Cm MATERIALS AND METHODS cartridges (Waters Associates. Milford. MA) previously prewashed with 10 ml 0.1% trifluroacetic acid. Cartridges were then washed with 10 ml 0.1% tri- Patients, Tumors, and Tumor Cell Lines. We studied patients entered in fluroacetic acid and rewashed with 10 ml 5% acetonitrile plus 0.1% trifluro institutional review board-approved lung cancer treatment protocols at the acetic acid, and immunoreactive ANF was then eluted from the Sep-Pak National Cancer Institute-Navy Medical Oncology Branch. National Naval cartridge with 3 ml 60% acetonitrile plus 0.1% trifluroacetic acid. The elutant Medical Center, from November 1, 1983, through November 31. 1988. The was dried overnight in a lyophilizer (Virus Co.. Gradiner. NY). patients were categorized as having small cell or non-small cell lung cancer, Different forms of human ANF were purchased from commercial sources which included adenocarcinoma. squamous cell carcinoma, large cell carci (ANF""-126 from Bachern. Torrance. CA: ANF1"2 I2h and ANF""-126 from noma, and bronchioalveolar carcinoma, and entered into separate therapeutic Peninsula Laboratories. Belmont. CA). Antibody directed against ANF was protocol studies (41-44). raised by covalently linking ANF to keyhole limpet hemocyanin conjugate The staging evaluation of patients with small cell lung cancer was described suspension (CalBiochem, CA) mixed with Freund's complete adjuvant in a prior publication (45). and following these staging procedures, the patients (GIBCO BRL) and injected into rabbits. Subsequent injections mixed with were classified as having either limited-stage or extensive-stage disease as incomplete Freund's adjuvant were given at 2-week intervals. Blood was previously defined (46, 47). obtained from rabbits 3 days after the second immunization and thereafter. The serum electrolytes, BUN, serum creatinine. serum osmolality, urine Dried elutants from processed cell lines were reconstituted in 0.5 ml RIA buffer electrolytes, and urine osmolality of SCLC patients were obtained prior to consisting of 0.01 M PBS containing 1% bovine serum albumin. 2% normal the initiation of chemotherapy, unless otherwise specified. The serum elec rabbit serum, and 0.01% thimerosal as preservative, pH 7.4. Stock aliquots of trolytes of the NSCLC patients were obtained prior to the tumor resection 10 mg/ml a-human ANF (Bachern) were stored at -20Â°C in 0.01 M PBS of the patients who were surgically treated. If multiple serum sodium values containing 1% bovine serum albumin. In this condition, the peptide retained its were available, the lowest value was selected for all patients. Patients with immunoreactivity for about 6 months. A portion of the reconstituted samples serum sodium values < 130 mmol/liter were classified as being hyponatremic (or standards; 0.1 ml) was incubated with 0.1 ml rabbit anti-human ANF serum (1. 3. 19. 21). (final dilution. 1:30.000) and 0.1 ml l25-I-a-human ANF (1300 Ci/mmol) The patients with SCLC initiated treatment with combination chemotherapy (Peninsula Laboratories. Belmont CA) for 18 h at 4Â°C.The bound ligands were with or without radiotherapy as previously defined (41, 43, 44). NSCLC separated from the free ones by the double-antibody method using goat anti- patients were treated with surgery, radiotherapy, or chemotherapy, singly or in rabbit IgG (ICN Biomedicals, Costa Mesa. CA) and 7.5% polyethelene glycol. combination. Tumor cell lines were established from biopsy tumor specimens The minimum detectable quantity was 1.27 fmol/tube (95% confidence), and according to techniques described before (42. 48, 49). the 50% binding intercept was 12 Â±2.07% pmol/tube (mean Â±SE. n = 6). The RNase Protection Assay. Tumor cell lines were grown in either ACL4 medium (49), RPMI-1640 |GIBCO, Bethesda Research Laboratories (BRL), intra- and interassay coefficients of variation (n = 10) were 7 and 10.7%, respectively. The antiserum had 100% cross-activity with rat ANF99"126, Grand Island, NY) with 5% heat-inactivated fetal calf serum (GIBCO BRL) or human ANF9" 126, human ANF102 l26, human ANF1"3'126, 30% with BNP, in RPMI-1640 HITES media (50) with 2-10% fetal calf serum. All media <1% with vasoactive intestinal protein, and none with angiotensin II. adreno- included I(X) units/ml penicillin and 100 ug/ml streptomycin (GIBCO BRL). corticotropic hormone (ACTH), AVP. ÃŸ-endorphin. met-enkephalin. neuro- Human right atria were obtained at the time of postmortem examination, frozen at -70Â°C.and minced on solid carbon dioxide. Tumor cell lines and atrial tissue tensin. oxytocin. somatostatin. and substance P. Arginine vasopressin and rabbit anti-human AVP serum were purchased were homogenized in 4 M guanidine isothiocyanate buffer using a Polytron (Kinematica GmbH. Lucern. Switzerland). Total cellular RNA was then pre from Amersham International, Buckinghamshire, England. The elutants from pared using the guanidium isothiocyanate and cesium chloride gradient method processed cell lines were reconstituted in 0.5 ml RIA buffer consisting of 0.050 and quantified by the absorbance at 260 nm (51). Size fractionation of RNA MPBS and containing 0.2% bovine serum albumin and 0.010 MEDTA. Stock aliquots of l(X) pmol/ml AVP were stored at -20Â°C in 0.050 MPBS containing samples on 0.8% agarose-0.66 M formaldehyde gels showed no significant degradation, as determined by comparison of ethidium bromide-staining in 0.2% bovine serum albumin. In this condition, the peptide retained its immu tensities of the 28S and 18S rRNA species. noreactivity for about 6 months. A portion of the reconstituted samples (or The AVP template was a Smal-Pvuil, 483-base pair, genomic fragment standards, 0.1 ml) was incubated with 0.1 ml rabbit anti-human AVP serum (final dilution. 1:30.000) and 0.1 ml (3-['25-I]iodotyrolsyl2)vasopressin (2000 containing portions of the second and third exons and the second intron (2). Ci/mmol)(Amersham International) for 24 h at 4Â°C.The bound ligands were with an EctiRl restriction site from the pGEM-3 (Promega Biotec. Madison, WI) sequence linked to the .V end of the Smti\ site. This EcoR\-P\-ull fragment separated from the free ones by the double-antibody method using goat anti- was subcloned into the Smal-EcoRl site of pGEM-3 transcription vector and rabbit IgG (ICN Biomedicals) and 7.5% polyethelene glycol. The minimum linearized with Ea>R\. The ANF template was a 200-base pair Pvu\l-Pxtl detectable quantity was 0.313 fmol/tube (95% confidence), and the 50% bind complementary DNA fragment including the 3'-coding and 3'-untranslated ing intercept was 0.87 Â±0.02% fmol/tube (;i = 3). The intra- and interassay sequences subcloned into the Pvull-Pstl site of pGEM-3 transcription vector coefficients of variation (n = 4) were 4.5% and 7.5%, respectively. (3) and linearized with PniH. Single-stranded "P-labeled AVP or ANF probes Statistical Methods. Trend tests for association between RNase and pellet were generated by Sp6 extension of AVP or ANF fragments subcloned into values were assessed using the Jonckheere test (53). Correlations between pGEM-3, followed by DNA template digestion with RQI-DNase according to sodium values and AVP peptide hormone levels and ANF peptide hormone instructions of the manufacturer (Promega). ANF- or AVP-specific probes levels were performed using Spearman rank correlation analysis. The signifi (200,000 cpm) were hybridized overnight at 56 or 62.5Â°C, respectively, to cance of differences in proportions was determined by the x2 test. The Kaplan- 30 ug total cellular RNA. Atrial natriuretic factor and AVP hybrids were Meier method was used to calculate the probability of survival as a function of digested with 14 and 28 ug in RNase A (USP). respectively, in 400 ml total time for a variety of factors (54), and the Mantel-Haenszel technique was used volume, for 30 min at 34Â°C,and the protected species were resolved by to compute the significance of the difference between the pairs of survival polyacrylamide gel electrophoresis (52). Signals were subjectivity quantified curves (55). Spearman rank coorelation analysis was used to associate RIA as strongly positive (++), positive (+), and negative (-). values with mRNA expression. Wilcoxan correlation was used to compare RIA Peptide Measurements in Tumor Cell Line Lysates. Approximately 0.5- values. 1.0 ml packed tumor cells was harvested during log phase growth and washed The Cox proportional hazards-modeling technique was used to identify twice in PBS at 4Â°C.The cells were suspended in 5 ml cold PBS and placed which factors, when simultaneously evaluated, may together affect survival in in boiling water for 15 min. The specimens were then homogenized for 1.5 min a statistically significant way (56). The resulting model parameters (/>,) were with a tissue tearer (Biospec Products. Barlesville. OK). The homogenates converted to relative risks by computing exp (h,), where exp (a) = 2.7183" (57).

The 95% confidence interval for the relative risk was computed as [exp (Â¿>,/.). proportional hazards modeling was performed to evaluate serum so exp (/>,/,)]. where b,, = b, - 1.96 (estimated SE (/>,)] and b,H = h, + 1.96 dium in conjunction with other previously established prognostic fac [estimated SE (/>,)]. The relative risk indicates the risk associated with dying tors [performance status, number of metastatic sites, and bilirubin while being in a greater risk category compared with that of being in a lower (45)]. The model in patients with extensive-stage SCLC and lower risk category. All P values are two-sided and denoted I'. sodium levels were independently associated with shortened survival (P = 0.012; relative risk. 1.31). The model in patients with both RESULTS limited- and extensive-stage SCLC. however, showed that hypo Clinical Data. Lung cancer patients (n = 263) were entered in natremia was not independently associated with shortened survival (P = 0.97; relative risk, 1.01). studies between November 1, 1983, and November 30, 1988. Of 133, 21 (16%) patients with SCLC presented had with hyponatremia Of the 21 patients with hyponatremia, 11 had tumor cell lines (serum sodium, <130 mmol/liter), in contrast to 0 of 130 (0%) established, and ANF and AVP mRNA expression studies and peptide patients with NSCLC (P < 0.0001). Of 87 extensive-stage SCLC analyses were performed. Ten of these 11 patients had extensive-stage disease, and patient IO (NCI-HI963) had limited-stage disease. Serum patients, 18 (21%) had hyponatremia in contrast to 3 of 46 (7%) of limited-stage patients (P = 0.033). sodium values for these hyponatremic patients ranged from 106 to 129 The 21 patients with SCLC who had hyponatremia lived a shorter mmol/liter. BUN and creatinine values were within normal limits for period of time than patients with normal serum sodium values at the all hyponatremic patients, except for patient 5 (Table 1). The serum time of presentation, but this difference did not achieve standard osmolality was inappropriately lower than the urine osmolality in all statistical significance (Fig. 1A; P = 0.08). However, the 3 patients 8 patients with available values. Urinary sodium values ranged from who were hyponatremic with limited-stage disease lived slightly < 10 to 201 mmol/liter. longer than the other patients with limited-stage disease. The 2 pa In order to determine the ANF and AVP mRNA expression and tients who lived the longest were treated on with the current combined peptide production in SCLC patients without hyponatremia. 8 exten sive-stage and 2 limited-stage SCLC patients with serum sodium modality trial with longer survival than we have previously observed, potentially explaining their prolonged survival (44). An analysis re values between 135 and 148 mmol/liters (median 139) throughout stricted to patients with extensive-stage SCLC showed that the 18 their clinical course and who had tumor cell lines available for mRNA patients who had hyponatremia lived a significantly shorter period of expression and peptide analysis were selected. These tumor cell lines time than the patients who did not have hyponatremia at the time of included 2 patients with limited-stage disease (patients 20 and 21 ) and presentation (Fig. IB; P = 0.012). A multivariate analysis using Cox 8 patients with extensive-stage disease, similar to the hyponatremic patients with SCLC. Ten NSCLC patients who had tumor cell lines available for ANF and AVP mRNA expression and peptide analysis were selected. This group consisted of 8 patients with serum sodium values between 135 and 143 mmol/liter, one patient with a value of 133 mmol/liter. and another with a value of 150 mmol/liter. The tumor Total Fail 112 104 O Sodium *I30 cell lines we chose to study from patients with NSCLC and patients 21 20 â€¢¿Sodium<130 with SCLC and normal serum sodium values grew at a rapid rate in order to provide adequate amounts of tumor cells for RIA analysis. RNase Protection Assay and RIA Data. Atrial natriuretic factor RNase protection assay data revealed that 9 of 11tumor cell lines from the patients with SCLC and hyponatremia had detectable ANF ex pression, 7 with strong signals (++) and 2 visible signals (+), and 2 cell lines had no detectable signal (-) (Fig. 2A). Cell lines from 7 of the 11 hyponatremic SCLC patients were shown to express AVP mRNA, er 5 cell lines with strong signals (++), 2 with visible signals (+), and 4 with no detectable signal (-) (Fig. 2B). All 11 cell lines from patients with SCLC and hyponatremia produced either ANF or AVP mRNA, o and 5 cell lines expressed both ANF and AVP mRNA. oc Radioimmunoassay results confirmed the presence of ANF or AVP peptide in all tumor cell lines with corresponding ANF and AVP mRNA expression (Table 2). The peptide hormone values >9 and 0.4 pmol/g for ANF and AVP, respectively, were more closely associated with mRNA expression. The peptide hormone levels in tumor cell lines tended to be highest in the cell lines from the 6 hyponatremic patients in which expression of only one of the 2 peptides was de tected. The ANF or AVP peptide levels detected by radioimmunoassay were usually higher in cell lines which had expression of only ANF or AVP compared to the ANF or AVP peptide levels in the 5 tumor lines that expressed both ANF and AVP mRNA, although this did not achieve standard statistical significance (P = 0.075 and 0.064. respec tively). The RNase protection assay of NCI-H711 mRNA produced SURVIVAL TIME IN MONTHS the strongest AVP signal, and the RIA value was nearly 1000-fold Fig. I. A, survival of patients with SCLC and serum sodium values il30 mmol/liter compared to patients with scrum sodium values < 130 mmol/liter (P = 0.081; B. survival more than all other cell lines studied. of patients with extensive stage SCLC and serum sodium values >I30 mmol/liter, com In the 10 cell lines from patients with SCLC and normal serum pared to patients with extensive stage SCLC and serum sodium values

Table I Chemistry values of hyponatremic paiients with cell lines available SerumPatient

no.123456789HI11CelllineNCI-H678NCI-H71INCI-HI061NCI-HI mL)81561!5(1147163125Crealinine(mg/100mLl0.70.70.60.81.31.00.Â«0.51.00.70.5OSM(mosM/kg)245240"250281246'246"248239mmol/L)1020069"16015<1092r201203985'

(192NCI-H1284NCI-HI

522NCI-HI6I8NCI-HI

788NCI-HI 876NCI-HI 963NCI-H208INa+(mmol/L)128126118120120''125124126122106129BUN(mg/HX) 'UrineOSM(mosM/kg)363922598"500573904463'894402552429'Na+( " Serum OSMand urine OSMand Na* values for NCI-H1061 were obtained I day after treatment began. h Serum Na value for NCI-HI284 was obtained 18 days before the other values were obtained. ' Serum and urine OSMand Na* values for NCI-HI6I8 were obtained 2 days after treatment began. ''Serum OSMfor NCI-H1876 was obtained 5 days after the other values were obtained. ' Serum OSMand urine OSMand Na* values for NCI-H208I were obtained 3 days after treatment began. signals (+). and 2 had undetectable signals (-). No cell lines from associated with serum sodium values of the patients from whom the SCLC patients with normal sodium levels demonstrated strong AVP cell lines were derived (P = 0.066, r = 0.12). mRNA signals, although 2 cell lines demonstrated visible AVP mRNA signals (Table 2, Fig. 35). Nine of the 10 tumor cell lines expressed DISCUSSION only one of the 2 peptides. Radioimmunoassay results confirmed the presence of ANF or AVP peptide in 7 tumor cell lines with corre Our patients with lung cancer prospectively studied with therapeu sponding AVP and ANF mRNA expression (Table 2). The peptide tic protocols confirmed previously reported differences in the inci hormone levels in tumor cell lines from patients with normal serum dence of serum sodium abnormalities between patients with non-small sodium values were generally lower than in those cell lines from cell and small cell lung cancer. None of 130 patients with NSCLC had hyponatremic patients which expressed only one of the 2 peptides hyponatremia, while 21 of 133 (16%) patients with SCLC had hy (Table 2). with the exception of NCI-H889. Immunoreactive ANF ponatremia, consistent with previous observations that patients with peptide levels were 30-fold higher in NCI-H889 than in any other cell NSCLC are much less likely to have SIADH than patients with SCLC line. The ANF RIA values in SCLC cell lines ranged from <3 to 10,000 pmol/g. The AVP RIA values in SCLC cell lines extended from Table 2 RNase protection assay and radioimmunoassay data <0.1 to 1400 pmol/g. The RIA values of both these peptides in the 20 SCLC cell lines varied >3000-fold. assayCell protection pelletANF In 3 tumor cell lines. RIA results showed the presence of immu- Patient noreactive peptide, while RNase protection assay results indicated no no.1234567g91011RNaseline(pmol/g)Afa*11031101923011231377<3390RIAAVP(pmol/g)0.614000.41.00.90.90.60.21.51.30.3SCLCAVPSCÃ•.C ANF mRNA expression. Atrial natriuretic factor immunoreactivity was de lowNCI-H678 cell lines from patients with tected in NCI-H660 and NCI-H 1450, but no ANF mRNA signal was ++NCI-H7I1 ++ detected. Arginine vasopressin immunoreactivity was detected in ++NCI-H106I ++ NCI-H 1304. but no AVP mRNA signal was detected. ++NCI-H ++NCI-H12841092 ++ ANF RNase protection assay data revealed that 2 of 10 cell lines ++NCI-H from the patients with NSCLC had visible ANF mRNA signals (+) +NCI-H16181522 ++ (Fig. 1A, Table 2), and none had detectable AVP signals (-) (Fig. 2B, +NC1-H1788 + +NCI-H Table 2). Cell lines with visible ANF mRNA signals (NCI-H810, 1876++NCI-H NCI-HI 155) also had markers of neuroendocrine differentiation, such 1963++NCI-H2081 ++Cell as bombesin-like immunoreactivity and L-dopa decarboxylase, as pre viously described (58). Radioimmunoassay results confirmed low or A/a*j cell lines from patients with normal undetectable ANF or AVP peptide in all NSCLC cell lines (Table 2). â€¢¿>131415161718192021NCI-H660+NCI-H735 There was a close association between ANF and AVP mRNA ex +NCI-H774 1<0. ++NCI-H889 10.4<0. pression and ANF and AVP peptide values in the tumor cell lines from ++NCI-HI the 30 patients with lung cancer (P = 0.0005 and 0.00035, respec 10.21.50.10.2N.D.NSCLC105+NCI-H +NCI-H1173 + tively). The association between ANF and AVP mRNA expression and 1304++NCI-H1450NCI-H peptide values was more closely associated with hyponatremia for AVP than for ANF. AVP expression and peptide hormone production 1672+NCI-H 1769 ++16<3661(XXX)<314151914N.D.0.3<0. was present in 7 of 11 tumor cell lines from patients with SCLC and hyponatremia, 2 of 10 from patients with SCLC and normal serum Na+22232425262728293031NCI-H810cell lines from patients with normal sodiums, and none often from patients with NSCLC. ANF expression +NC1-H838NCI-H and peptide hormone production was present in 9 of 11 tumor cell i 155+NCI-H 10.40.20.2<0.10.4<0. lines from patients with SCLC and hyponatremia, 8 of 10 from pa 1264NCI-H tients with SCLC and normal serum sodiums, and 2 of 10 from 1299NCI-H patients with NSCLC. The AVP peptide hormone level in 30 tumor 1334NCI-H 1355NCI-H cell lines from the lung cancer patients was moderately associated 1373NCI-H with the serum sodium values (P = 0.0026, r = 0.28). In contrast. 1437NCI-H 1<0.1 1581<3g<3<3<3<34<3<3<30.10.1<0. ANF peptide hormone level in 30 tumor cell lines was only marginally 70

Hyponatremia was more common in our patients with extensive- HYPONATREMIA stage disease (21%) compared to 6% of patients with limited stage disease (P = 0.033). Although earlier studies have shown that hy- IIXIII ponatremia is more common in patients with extensive-stage disease

820 bp - than in patients with limited-stage disease, this difference has usually 520 bp Â«63bp not achieved standard statistical significance (P < 0.05) (20, 21, 61).

298 bp-

SCLC NSCLC 220 bp- â€¢¿227bases - 200 bases o S o Ã•8g jn CL i< i i 1 i I 154 bp- => o o o u O U U (J O O I I z z z z z z z z z z 620 bp- 520 bp - 453 bp-

298bp- I^B

- 227 bases HYPONATREMIA 220bp- ' - 200 bases

o.> "z< uuuuuuuuouuu-i^._i^._i_i_i^._i_iâ€” â€”¿ u < Ã• zzzzzzzzzzzz z 154bp- 1026 bp - B 620 bp -â€¢ 520 bp - 453 bp-

298 bp-

220 bp-

SCLC NSCLC

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position in order to emphasize that it lacked any detectable signal. EXON Z EJCON3 -164 bp- â€”¿BOtw-

Fig. 3. RNase protection assay for ANF mRNA (A) and AVP mRNA (B) in SCLC and NSCLC tumor cell lines from patients with normal serum sixlium levels. A, a radiolabeled (1). The absence of hyponatremia in patients with NSCLC is poten single-stranded ANF probe was hybridized to 30 ug total cellular RNA from SCLC tumor tially explained by the fact that NSCLC cell lines only infrequently cell lines or 30 ug total cellular RNA from NSCLC tumor cell lines or 1.0 ug total human right atria RNA. Digestion with RNase A resulted in a delectable signal of 200 bases. Left produce peptides which can alter serum sodium values. None of the 10 ordinate, positions for single-stranded nucleotide size markers; right ordinate, undigested NSCLC cell lines produced AVP mRNA or peptide, and only 2 of 10 and digested probe sizes. Human right atria protected the predicted species of 200 bases, tumor cell lines produced ANF mRNA. Our finding that 16% of and NCI-H526 served as the negative control. B, a radiolabeled single-stranded AVP probe of 483 bases was hybridized to 30 ug total cellular RNA from either SCLC or NSCLC SCLC patients had hyponatremia is in agreement with collective tumor cell lines. Digestion with RNase A resulted in detectable signals of IH4 and 80 studies which show that approximately 14% (range 7-35%) of SCLC bases. Left ordinate, positions for single-stranded nucleotide size markers; right ordinate, protected probe sizes. RNA from tumor cell line NCI-H378 protected the predicted patients have clinically evident hyponatremia or SIADH at the time of protected species of approximately 183 bases (exon 2) and 80 bases (exon 3), and presentation (13-15, 19-21, 59, 60). NCI-H526 served again as the negative control. 71

The data generated in this study by RIA of SCLC cell pellets provide prohormone. An enzyme processing defect in a pair of related pro- insight into why patients with limited-stage disease do not always hormones produced ectopically in small cell lung cancer was been have a reduced incidence of hyponatremia as compared to patients previously identified. Proopioimelanocortin and pro-ACTH are not with greater tumor burden (extensive-stage disease) in other reports. processed to mature ACTH in SCLC cell lines (63), and a similar In SCLC cell lines, the ANF and AVP peptide values determined by defect may exist in the processing of ANF in NCI-H889. Both the RIA varied >3000-fold for both peptides. Concentrations of 2 other mutation and processing defect could explain the high levels of ANF peptide hormones, calcitonin and bombesin, have been shown to mRNA expression and ANF immunoreactivity in NCI-H889 without vary >300- and 2000-fold, respectively, in different SCLC cell lines resulting in abnormal serum sodium levels. Atrial natriuretic factor (48, 62). This suggests that the capability of the tumor to produce sequencing and processing studies of NCI-H889 are underway to peptide hormones varies greatly from patient to patient. Therefore, the investigate these possibilities. total amount of ANF and AVP peptide produced by SCLC is a function Radioimmunoassay detected ANF or AVP immunoreactivity in four of both intrinsic peptide hormone production as well as tumor burden. of our lung cancer cell lines in which RNase protection assay did not The 18 patients with extensive stage SCLC who had decreased detect ANF or AVP mRNA signal. NCI-H660, NCI-H1450, and NCI- levels of serum sodium lived a shorter period of time than patients H838 had no detectable ANF mRNA, but had ANF immunoreactivity. without hyponatremia (P = 0.012), and decreasing levels of sodium NCI-HI304, had no detectable AVP mRNA but had AVP immunore were shown to be significant by association with minimal risk of death activity. Potential explanations for this disparity include nonspecific by Cox model (P = 0.012). This agrees with the majority of studies of immunoreactivity detected by the polyclonal antiserum or the detec prognostic factors in patients with SCLC, which have shown that tion of related peptides which cross-react with the antibody such as hyponatremia is associated with shortened survival in multivariate BNP, which has 30% cross-reactivity with the ANF antibody. Studies analyses (36-38). A potential explanation why patients with hy are underway to determine whether ectopie production of BNP occurs ponatremia live a shorter period of time is that AVP can cause hy in small cell lung cancer. ponatremia and has been shown to function as a growth factor in small Several studies have linked hyponatremia in patients with intracra- cell lung cancer cell (39, 40). Therefore, AVP may increase the rate of nial disease to elevated plasma ANF levels. Patients with intracranial tumor growth and lead to a more rapid death of the patient. Although disease, which include subarachnoid and intracerebral hemorrhage, we found an association between low sodium and shortened survival, Creutzfeldt-Jakob disease, and glioblastoma multiforme developed we did not find that patients who made AVP lived a shorter period of elevated plasma ANF levels, followed by natriuresis, volume deple time than patients who made ANF. tion, and occasional hyponatremia (64-66). These studies have indi In our examination of mRNA expression in tumor cell lines from cated that the volume depletion and hyponatremia associated with hyponatremic SCLC patients, we have shown that all 11 cell lines these intracranial abnormalities do not appear to be associated with produced detectable levels of either AVP or ANF mRNA and peptide AVP but rather with ANF (65, 66). Therefore, it appears that elevated or both (Table 2). Radioimmunoassay for AVP and ANF peptides was plasma ANF levels are associated with hyponatremia in patients with performed to confirm peptide production. In addition. RIA results are intracranial disease as well as with hyponatremia in patients with easier to quantify than RNase protection assay results and reflect more small cell lung cancer. accurately the amount of peptide produced. Cell pellet RIA values for In the original description of SIADH by Bartter and Schwartz (67), ANF and AVP were closely associated with ANF and AVP mRNA they proposed three potential factors which could explain sodium loss expression. We were unable to study plasma levels of ANF and AVP with SIADH (67). These three factors included: (a) a factor causing because, in this retrospective analysis, nearly all patients were de suppression of aldosterone secretion resulting from an increase of ceased; therefore, plasma was unavailable for analysis. However, extracellular fluid volume, (b) a factor causing an increase in the these hormones have been studied in patients with SIADH and lung filtered load of sodium resulting from an increase in the glomerular cancer, and all patients but one had elevated ANF and AVP immu- filtration rate, and (c) a factor causing the suppression of tubular noreactivity in the plasma ( 16, 22, 23, 35). In this series of 11 cell lines reabsorbtion of sodium in response to expansion of the extracellular from patients with SCLC and hyponatremia, the most common find fluid volume. The functions of ANF are consistent with these three ing was ectopie production of both ANF and AVP mRNA and immu- proposed factors explaining the salt loss with SIADH. Atrial natri uretic factor suppresses aldosterone secretion (68-71). Glomerular noreactive peptide. This potentially explains why plasma levels of filtration rate is increased by ANF (72-75), and ANF inhibits sodium both ANF and AVP are commonly elevated in patients with SCLC and into the renal tubules (76-79). From this evidence, we believe that SIADH. Our findings demonstrated a modest association between AVP pep ectopie secretion of ANF by SCLC tumor cells may cause hyponatre tide production in tumor cell lines and hyponatremia in patients with mia and contribute to the abnormalities of SIADH. The conclusive SCLC (P = 0.0026, r = 0.28) and a less significant association proof that ANF can cause hyponatremia awaits the results of an between ANF peptide production and hyponatremia (P = 0.066. ongoing study of ectopie production of ANF and AVP in untreated r2 = 0.12). There may be a threshold of ANF production that must lung cancer patients (80). be surpassed before it lowers the serum sodium concentration. This is supported by the finding that 3 of the 4 patients with cell lines ACKNOWLEDGMENTS which had ANF RIA values of >IOO fmol/g and without AVP pro duction were hyponatremic. The one patient with values of >IOO We wish to acknowledge Drs. E. Sausville and J. F. Battey for providing the fmol/g in her tumor cell line, and normal serum sodium values AVP genomic clone used to generate the vectors used in the RNase protection (138 mmol/L) before treatment and throughout her course, had the assays. strongest RNase protection assay signal and highest RIA values in her tumor cell line (NCI-H889). Possible explanations for the ectopie REFERENCES production of large quantities of ANF without the patient developing 1. Bunn, P., Jr.. and Ridgeway, E. Paraneoplastic syndromes in cancer. In: V. T. Devita, abnormally low serum sodium levels include the inability of ANF to S. A. Rosenberg, and S. Hellman (eds.). Cancer Principles and Practice of Oncology, pp. 1896-1940. Philadelphia: J. B. Lippincott Company, 1989. alter serum sodium levels, a mutation in the protein-coding portion of 2. Sausville, E., Carney, D,, and Battey, J. 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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1993 American Association for Cancer Research. Atrial Natriuretic Factor and Arginine Vasopressin Production in Tumor Cell Lines from Patients with Lung Cancer and Their Relationship to Serum Sodium

Andrew J. Gross, Seth M. Steinberg, J. Garrett Reilly, et al.

Cancer Res 1993;53:67-74.

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