©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of a Protein That Permits Early Detection of Lung Cancer
IDENTIFICATION OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN-A2/B1 AS THE ANTIGEN FOR MONOCLONAL ANTIBODY 703D4 (*)

(Received for publication, November 13, 1995; and in revised form, February 17, 1996)

Jun Zhou James L. Mulshine Edward J. Unsworth Frank M. Scott Ingalill M. Avis Michele D. Vos Anthony M. Treston (§)

From the Biomarkers and Prevention Research Branch, National Cancer Institute, Rockville, Maryland 20850-3300

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have reported that a mouse monoclonal antibody, 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer-based C(18)-like, and analytical C(4) reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was >95% pure by Coomassie staining. The NH(2) terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C(4) high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis.


INTRODUCTION

Lung cancer is the most frequent cause of cancer death of both men and women in the United States, accounting for one in three cancer deaths(1) . In the last 30 years, cancer-related survival of this disease has improved minimally. Successful treatment of this disease by surgical resection and drug chemotherapy is strongly dependent on identification of early-stage tumors. A conceptually attractive early detection approach is to establish the presence of a cancer by evaluation of shed bronchial epithelial cells recovered in the sputum. Cytological approaches to evaluate precancerous cytomorphologic changes in the exfoliated bronchial epithelium have been reported(2) . However, early diagnosis clinical trials using combination chest x-ray and conventional sputum cytology have not shown any decrease in cancer-related mortality(3) . In 1988, Tockman et al.(4) reported a sensitive method for early lung cancer detection by immunostaining exfoliated cells in sputum samples with two lung cancer-associated monoclonal antibodies. The mouse monoclonal IgG antibodies used in that study were designated 624H12 and 703D4. 624H12 has been shown to detect an onco-fetal antigen that is the difucosylated Lewis^x structure(5) . The antigen for 703D4 was unknown. In an analysis of the contribution of the individual monoclonal antibodies to early detection of lung cancer, 703D4 alone identified 20 of the 21 detected true positive cases(4) .

703D4 was developed by immunization using a whole tumor cell extract coupled to keyhole limpet hemocyanin, and selection was based on discrimination among lung cancer histological subtypes. Preliminary studies showed that the 703D4 antibody recognized a protein expressed by most non-small cell lung cancer (NSCLC) (^1)cells(6) . Immunoprecipitation defined a protein of molecular mass of approximately 31 kDa. Since 703D4 antibody demonstrated the ability to selectively detect changes related to the development of cancer in shed bronchial epithelium from the proximal airways, we have used a biochemical approach to purify 703D4 antigen to determine its identity and explore its relationship to early lung cancer detection.


EXPERIMENTAL PROCEDURES

Electrophoresis and Western Blotting

To analyze the antigen-purification steps, an aliquot of the starting material and of each of the fractionations described below (ion exchange, IEF, and HPLC) was assayed by either Tris-Tricine or Tris-glycine SDS-polyacrylamide gel electrophoresis (PAGE). Aliquots were freeze-dried and reconstituted or diluted directly in either Tris-glycine sample buffer containing 5% mercaptoethanol or Tricine sample buffer and subjected to electrophoresis on a 4-20% Tris-glycine or 10-20% Tricine gel (NOVEX, San Diego, CA). Gels were fixed, stained with colloidal Coomassie Brilliant Blue G-250(7) , and photographed. Proteins on duplicate gels were electrophoretically transferred to PVDF membrane at 30 V for 1.5-2.0 h, blocked overnight at 4 °C with 1% bovine serum albumin in phosphate-buffered saline, and immunoblotted using antibody 703D4. 703D4 is an IgG2b(k) monoclonal antibody(6) . The antibody was affinity purified from mouse ascites using a Protein A Sepharose column and a glycine NaCl/citrate gradient. The bound antibody on the Western transfer PVDF membranes was detected using direct binding of radioiodinated staphylococcal Protein A. Blots were imaged on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and on Kodak XAR and XRP films.

Preparation of Cellular Subfractions

Human tumor cell lines, including the NSCLC cell lines NCI-H720 (carcinoid) and NCI-H157 (squamous) used for antigen purification, were grown in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% fetal calf serum at 37 °C and 5% CO(2). The cells were harvested and washed twice with ice-cold Dulbecco's phosphate-buffered saline (pH 7.4), resuspended in MES buffer (17 mM MES (pH 7.0), 20 mM EDTA, 250 mM sucrose, with 50 µg/mL leupeptin, 300 µg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride), and homogenized in a hand-held homogenizer. Trypan blue exclusion was employed to ensure greater than 90% cell lysis following homogenization. The lysates were transferred to Beckman polyallomer centrifuge tubes and subjected to centrifugation at 150,000 times g for 60 min using a Beckman XL90 ultracentrifuge and SW41 rotor. The pellet containing the membrane and nuclear fractions was retained, and the cytosolic supernatant was discarded(8) .

The pellets were resuspended in extraction buffer (0.15 M NaCl, 10 mM Tris, pH 7.4, 5 mM EDTA) containing 1% Tween 20 and the anti-protease mixture described above. The samples were incubated on ice for 1 h, with frequent vortexing, and subjected to centrifugation at 16,000 times g for 20 min. The supernatant was then diluted 3-fold with deionized water and adjusted to pH 6.5 for ion-exchange chromatography.

Ion-exchange Chromatography and Liquid Phase Isoelectric Focusing

A Dupont Bio Series WAX (weak anion exchange) column (MacMod, Chads Ford, PA), equilibrated with Tris-HCl, pH 6.5, was used. Detergent-solubilized proteins were pumped through the column at 2.0 ml/min. 703D4 immunoreactive material was eluted in the unbound material from this column in the presence of 50 mM NaCl, and the anti-protease mixture described above was added.

The unbound fractions positive for 703D4 antigen were pooled and freeze-dried, resuspended to a final volume of 45 ml with 4 M urea containing 3% CHAPS, 10% glycerol, and 0.8% Ampholine, pH range 3-10 (Bio-Rad). This protein-ampholyte mixture was loaded into a chilled Rotofor preparative isoelectric focusing (IEF) apparatus (Bio-Rad) which was operated at a constant 12 watts. One hour after the maximum voltage was reached, usually 1200 V, fractions were harvested by vacuum collection. Run time was approximately 4 h. pH values were determined for the 20 fractions which were harvested. 703D4 antigen was concentrated in fractions with pH 8-9.5. The three most positive fractions from each IEF run were pooled for HPLC purification.

Reversed-phase HPLC

All organic solvents used were HPLC grade (Burdick & Jackson, Muskegon, WI). The preparative IEF fractions positive for 703D4 antigen were diluted 2-fold with 18 megaohm water, acidified with a final concentration of 1% trifluoroacetic acid (TFA) (Pierce), and applied to a 10 mm times 10 cm Poros® perfusion polymeric analytical C(18)-like R2 column (PerSeptive Biosystems, Framingham, MA) which was equilibrated with 5% acetonitrile, 0.1% TFA. The protein was eluted using a 15-min linear gradient proceeding from 5% acetonitrile, 0.1% TFA to 100% acetonitrile, 0.1% TFA at a flow rate of 10 ml/min. Fractions of 2.5 or 5 ml (15-30 s) were collected after a 2.0-min wash. Next, the positive fractions (2.5-5.0 ml, 40% acetonitrile) were diluted 5-fold with water, 0.1% heptafluorobutyric acid (HFBA) (Pierce) and applied to another Poros polymeric R2 column equilibrated with 5% methanol, 0.1% HFBA. The protein was eluted with a 15-min linear gradient from 5% methanol, 0.1% HFBA to 100% methanol, 0.1% HFBA at a flow rate of 10 ml/min. The 703D4 antigen eluted at approximately 80% methanol.

As the last stage in the purification, the positive fractions were applied to a 2.1 mm times 25 cm Vydac analytical C(4) column (Vydac, Hesperia, CA), which was equilibrated with 20% acetonitrile, 0.1% TFA, and the protein was eluted at a flow rate of 0.2 ml/min with a triphasic linear gradient from 20% acetonitrile to 30% acetonitrile over 20 min, to 45% acetonitrile over 75 min (0.2%/min), then to 90% acetonitrile over 20 min. Fractions of 0.4 ml (2 min) were collected.

Digestion and Protein Sequencing

Several failed attempts at obtaining NH(2)-terminal amino acid sequence information, both from SDS-PAGE blotted material and directly from freeze-dried fractions at the last C(4) HPLC step, indicated that the NH(2) terminus of the purified protein was blocked. Therefore a CNBr digestion was employed to obtain the internal sequence. The purified protein, freeze-dried after the C(4) HPLC fractionation, was cleaved under nitrogen with 0.15 M CNBr (Fluka) in 70% formic acid at room temperature for 24 h(9) . The resulting peptides were separated by 16% Tricine SDS-PAGE and electroblotted onto PVDF membrane. The peptides were visualized using Ponceau S, and bands were excised for Edman degradation sequence analysis on an Applied Biosystems model 477A (Foster City, CA). Some proteins were also sequenced on an Applied Biosystems 494A. The amino acid sequence obtained was compared to known sequences in the SwissProt data base using PepScan (PE/SCIEX, Thornhill, Ontario, Canada).

Tissue Culture and Cell Treatment

Primary cultures (first or second passage) of human bronchial epithelium cells were grown in BEGM (Clonetics, San Diego, California) or Keratinocyte-SFM (Life Technologies, Inc., Gaithersburg, MD). To obtain stationary phase cells, confluent cells were maintained in BEGM for 4 days. We obtained the human adeno-12-SV40-transformed bronchial epithelium cell line (IB3-1)(10) . It was grown in BEBM with 5% fetal calf serum. Stationary phase cells were obtained by maintenance in BEBM with 5% fetal calf serum for 4 days after the cells reached confluence. NSCLC cell lines NCI-H157, HTB58 (squamous) and NCI-H23 (adenocarcinoma) were grown in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 5% fetal calf serum. To obtain stationary phase cells, confluent cells were maintained in RPMI 1640 medium with 5% fetal calf serum for 4 days. For all cell lines, log phase cells were harvested at 70-80% confluence.

Isolation of Total Cellular RNA and Northern Analysis

Medium was removed from the cells, and they were washed once in phosphate-buffered saline. Nonadherent cell lines were harvested and washed by centrifugation (800 times g, 10 min). RNA was extracted with guanidine isothiocyanate/2-mercaptoethanol and purified by ultracentrifugation as described previously(11) . After ultracentrifugation, the RNA pellet was resuspended in water, ethanol-precipitated in the presence of 0.3 M sodium acetate, and pelleted by centrifugation. The dried pellets were redissolved in water, and 10 µg of total cellular RNA from each of the tumor cell lines, normal lung, and normal bronchial epithelium primary cultures were used for Northern blot analysis. The RNA was resolved using a 1% agarose-formaldehyde gel with 0.2 M MOPS, 0.05 M sodium acetate, 0.01 M EDTA as the running buffer. The RNA was transferred passively to nitrocellulose membrane, hybridized, and washed. Autoradiography was performed according to standard techniques.

Northern analysis was carried out using probes prepared by random priming of inserts gel-purified from restriction endonuclease digests of plasmids containing full-length cDNAs for heterogeneous nuclear ribonucleoprotein (hnRNP)-A2. Approximately 1 times 10^6 cpm/ml of probe was used for each Northern analysis.

Reverse Transcription-PCR and a Southern Blot Analysis

Reverse transcription was performed with 0.2 µg of DNase-treated total RNA using the Superscript amplification kit according to the manufacturer's protocol (Life Technologies, Inc.). The resulting cDNA was subjected to 30 or 35 cycles of polymerase chain reaction (PCR) on a Perkin-Elmer GeneAmp PCR system 9600. The primers designed for the amplification were: 5`-GAGTCCGGTTCGTGTTCGTC-3` and 5`-TGGCAGCATCAACCTCAGC-3`. These primers were selected using Lasergene software by DNA-Star (Madison, WI), and were chosen to span a site of alternate exon utilization (36 nucleotides) which generates the hnRNP splice forms A2 and B1. The resulting amplified DNA was analyzed by electrophoresis on a 2.0% NuSieve agarose gel. DNA was then transferred to a nitrocellulose filter, hybridized, washed, and exposed to film as described previously(10) . Southern blot analysis was carried out with a P-end-labeled 20-nucleotide antisense oligonucleotide, which hybridizes with a sequence common to both hnRNP-A2 and -B1.


RESULTS

Biochemical Characterization of 703D4 Antigen

Preliminary data showed a wide range of expression of the 703D4 antigen in many NSCLC cell lines as judged by a solid phase radiobinding assay. All results shown are for purification steps using cell line NCI-H720, which grows rapidly as floating clumps of cells in culture medium containing 5% fetal bovine serum, allowing high cell density. After the methods were developed, an identical protocol was followed to purify the antigen from the original immunogen cell line, NCI-H157. 703D4 immunoreactivity at all stages of the purification was detected by SDS-PAGE followed by immunoblot analysis, as attempts to scale up our previously reported immunoprecipitation technique (6) were not successful.

Western blot analysis of crude extract under both reducing and nonreducing conditions revealed a major band with a molecular mass of approximately 31.5 kDa on NOVEX 10-20% Tricine gels or 34 kDa reduced on NOVEX 8-16% Tris-glycine gels (determined by comparison with Bio-Rad SDS-PAGE prestained standards). Only a single major immunoreactive protein was identified, although in the later stages of purification an apparent disulfide-linked homodimer appeared which could be removed by reduction with 2-mercaptoethanol, and at the final HPLC steps a minor band of slightly higher M(r) was identifiable (M(r) = 33,000 on Tricine gels, 35,000 on Tris-glycine gels) (Fig. 2C). Our original immunoprecipitation analysis identified similar molecular mass proteins (major band approximately 32.5 kDa, minor band approximately 34 kDa) using linear 10% acrylamide gels(6) .


Figure 2: C(4)-reversed phase HPLC purification of 703D4 antigen. 703D4 immunoreactive fractions from methanol/HFBA were injected onto a 2.1 mm times 25-cm Vydac C(4) column, which was eluted with a triphasic gradient of acetonitrile in 0.1% TFA. The central region of the chromatogram, from 33-48% acetonitrile is shown (A). Gradient and UV absorbance are denoted as in Fig. 1. Fractions were analyzed by SDS-PAGE as described for Fig. 1, except Tris-Tricine gels were used. B, the colloidal Coomassie Blue-stained gel; C, the Western blot. Migration of prestained protein standards (sizes in kDa) are indicated on the right.




Figure 1: Polymeric reversed phase HPLC purification of 703D4 antigen. A 10 mm times 10 cm Poros perfusion polymeric R2 column was equilibrated with 5% acetonitrile, 0.1% TFA (A) and 5% methanol, 0.1% HFBA (D). Protein was eluted with a gradient of 5-100% acetonitrile (A) and 5-100% methanol (D) at a flow rate of 10 ml/min. Gradient is indicated by a dashed line and UV absorbance (214 nm) by a solid line. A portion (3%) of each fraction was run on two identical Tris-glycine SDS-PAGE gels. One gel was stained with colloidal Coomassie Blue (C and F) and the other transferred to PVDF for reaction with 703D4 antibody (B and E). Migration of prestained protein standards are shown on the right. The major 703D4 immunoreactive protein eluted in fractions 15-16 (B) and fractions 34-35 (E).



Simple subcellular fractionation analysis of 703D4 antigen distribution according to the method of Krajewski et al.(8) showed that, except for a cytosolic supernatant, all membrane-bound fractions including the crude nuclear pellet had immunoreactive protein (data not shown). These data parallel our immunohistochemical characterization of 703D4 antigen expression in fixed cells, which showed binding to perinuclear and membrane-bound cytosolic sites(16) . The antigen in a NCI-H720 subcellular fraction containing nuclei and membrane-bound proteins could be solubilized by gentle extraction with either nonionic detergents such as Tween 20, Nonidet P-40, and Triton X-100 or ionic detergent such as 1% SDS.

Weak anion exchange chromatography of crude detergent-solubilized proteins at pH 6.5, 7.5, and 8.5 indicated all the immunoreactivity of the crude tumor cell extract was eluted in the unbound fraction in the presence of low (50 mM) salt. When the crude antigen was subjected to preparative IEF under denaturing conditions (4.0 M urea) the immunoreactivity appeared in fractions with pH 8-9.5.

Purification of 703D4 Antigen

The protein identified by 703D4 was isolated from NCI-H720 and -H157 cells by a six-step procedure. The first steps were carried out rapidly to prevent degradation of the target molecule. Using a variety of protease inhibitors or reducing agents, we were able to reduce but not eliminate antigen degradation. To prevent degradation during the SDS-PAGE and Western blot analysis of each fractionation step, the bulk of the material was stored frozen at -30 °C during the analysis.

A typical purification commenced with 5-10 ml of packed cells, washed with phosphate-buffered saline to remove serum proteins present in the cell culture medium. The initial step was subcellular fractionation to remove cytosolic proteins, followed by gentle nonionic detergent solubilization of the membrane-containing fraction. The detergent-solubilized fraction was then diluted to lower the salt concentration to 50 mM and injected onto the weak anion-exchange column. Studies with weak and strong anion and cation exchange resins demonstrated tight binding to cation and strong anion exchange matrices but poor recovery of immunoreactive material (results not shown). We therefore used the weak anion exchange resin to remove a significant portion (approximately 75%) of protein to prevent loss of 703D4-immunoreactive protein through co-precipitation at the IEF step. In the presence of 50 mM salt, 703D4 antigen eluted with the unbound fraction which was freeze-dried and redissolved in a denaturing buffer for preparative IEF. IEF concentrated the immunoreactive protein into a basic region of the pH gradient. Protein from several batches of IEF was pooled at this point for HPLC purification.

The HPLC chromatograms from the next stages of this procedure are shown in Fig. 1and Fig. 2. Attempts to remove the ampholytes and urea after preparative IEF by molecular sieve chromatography or direct injection onto silica-based reversed phase HPLC matrices resulted in precipitation of the target protein and loss within the column matrix. The Poros® macroporous polymeric analytical R2 column rapidly and efficiently desalted the antigen from the urea/ampholyte mixture and simultaneously separated 703D4 immunoreactivity from the bulk of the other proteins in the mixture (Fig. 1). Our HPLC procedures utilize mobile phases usually applied for peptide analysis and/or purification, but these conditions proved very effective for this protein purification. The chromatographically ``weaker'' organic modifier (methanol) used with the more lipophilic ion-pairing agent (HFBA) resulted in a distinctly different mobility of the 703D4 antigen compared with that in the acetonitrile/TFA mobile phase. These conditions also provided selectivity for removal of other proteins present in the sample. The two solvent systems resulted in significantly greater purification of the target molecule than either solvent system alone.

Analytical C(4) reversed-phase HPLC with an acetonitrile gradient containing 0.1% trifluoroacetic acid was used as the final purification step. 2.5-5.0 ml of 703D4-immunoreactive fractions from the methanol/HFBA polymeric R2 column was diluted 5-fold with water, 0.1% TFA, injected onto a Vydac C(4) column, and eluted with a slow gradient of acetonitrile (0.2%/min). Immunoblotting analysis of the C(4) fractions revealed two immunoreactive proteins with distinct sizes as determined by SDS-PAGE (Fig. 2C). The lower and later eluting protein is the principal 703D4 immunoreactive antigen and was greater than 95% pure as determined by Coomassie staining of the SDS-PAGE gel. The immunoreactivity paralleled the Coomassie staining intensity for both the major and minor bands, demonstrating homogeneity of the purified proteins (Fig. 2, B and C).

Determination of recoveries of immunoreactivity at each step could not be made using a Western blot analysis method; however, we estimate an overall yield of 5-10% for the six-step procedure. Final yield of the principal immunoreactive protein from a typical purification, determined from NH(2)-terminal Edman sequence yield, was 200 pmol. We estimated overall purification from the total protein in the starting material and the final yield of purified antigen. This yield implies an approximately 25,000-50,000-fold overall purification, although as stated the Western blot detection method did not allow for an accurate quantitation of any loss of immunoreactive material during the purification. Table 1shows the approximate fold purification for each step of the procedure, averaged from several typical purifications.



Amino-terminal Sequencing of CNBr Fragments of 703D4 Antigen

Several attempts to obtain amino-terminal sequence of purified 703D4 antigen were not successful, including direct sequencing from the C(4) HPLC fractions. The major immunoreactive protein, that is, the later eluting, lower M(r) band on SDS-PAGE of the analytical C(4) purification step, was therefore concentrated by freeze-drying the peak fractions and cleaved using 0.15 M CNBr in 175 µl of 70% formic acid (9) . After a 24-h reaction in the dark at 25 °C, the CNBr digest was diluted with 1.0 ml of water and freeze-dried. The peptides were solubilized directly in Tricine loading buffer. Four bands were separated and visible after Tricine SDS-PAGE on a linear 16% gel, electroblotting onto PVDF membrane, and staining with Ponceau S or Coomassie Blue (Fig. 3). All four bands were subject to 12 cycles of Edman degradation on an ABI 477A using the standard ABI protocol for blotted proteins. The sequences revealed were: AARPHSIDGRVV (27- and 13-kDa bands), QEVQSSRSGRGG (15-kDa band), and EREKEQFRKLFI (4-kDa band). Each amino acid was the major or only amino acid called by the automated sequencer at each cycle.


Figure 3: 16% Tricine SDS-PAGE analysis of products of CNBr digestion of purified 703D4 principal antigen. The left lane contains the purified antigen before digestion. The arrows indicate the four major bands which were subjected to amino-terminal Edman sequencing.



A search in the SwissProt sequence data base of each of these sequences identified a single gene product. The sequences, and the size of the cyanogen bromide digestion products, are consistent with the major 703D4 antigen being hnRNP-A2. Fig. 4shows these sequences aligned with the translated cDNA sequence of hnRNP-A2/B1, which includes a previously reported 36-nucleotide (12-amino acid) exon close to the protein amino terminus that is specific for hnRNP-B1. The 4-kDa CNBr fragment sequence crossed this site of alternate exon splicing, demonstrating that the major antigen is hnRNP-A2. As expected for CNBr-generated fragments, each sequence is immediately COOH-terminal to a methionine residue in the predicted sequence. The two identical sequences obtained for the 27- and 13-kDa bands and the presence of faint bands not readily visible in Fig. 3implies incomplete CNBr digestion, possibly due to oxidized methionines in the freeze-dried protein. For a parallel purification of 703D4-immunoreactive protein from the original immunogen cell line NCI-H157, an identical sequence was obtained from the CNBr-generated 13-kDa band (AARPHSIdgRVV) and some confirmatory sequence was obtained from the 15-kDa band (amino acids in upper case represent the primary amino acid in each cycle, and lower case letters denote amino acids identified as the secondary calls). CNBr digestion resulted in loss of immunoreactivity with 703D4, indicating that the antibody probably recognizes a conformational epitope. Several recent reports demonstrate that monoclonal antibodies raised to intact proteins commonly recognize conformational epitopes(12, 13) .


Figure 4: NH(2)-terminal sequences of peptides isolated from CNBr digest of purified 703D4 antigen. The NH(2)-terminal amino acid sequences and approximate molecular masses of the CNBr cleavage fragments of the purified 703D4 major (hnRNP-A2) and minor (hnRNP-B1) antigens are indicated. Arrows indicate the positions of methionines within the protein, and the hatched area indicates the site of the alternately spliced exon differentiating hnRNP-A2 from -B1. All peptides which were not recovered are too small to be resolved from the migration front of the Tricine SDS-PAGE gel (<2.5 kDa).



The last step in the purification of the 703D4 antigen resolved a second immunoreactive band of slightly higher molecular size, and parallel immunoreactivity (judged by a comparison of the Coomassie and immunostaining intensities). A CNBr digestion was carried out on C(4) HPLC fractions (pooled from three separate purifications) containing the minor immunoreactive band which eluted slightly before the hnRNP-A2. The CNBr digest yielded two principal Coomassie-stained bands after Tricine SDS-PAGE. The approximately 5-kDa band was subjected to NH(2)-terminal Edman degradation on an Applied Biosystems 494A and yielded a sequence EKTKEtVPlerKkrE (as above, amino acids in upper case represent the primary amino acid in each cycle, and lower case letters denote amino acids identified as the secondary calls). This sequence is identical to that of the hnRNP-B1 CNBr fragment which includes the 12-amino acid insertion not present in hnRNP-A2. A second lower level sequence present in the same sample was consistent with hnRNP-A2, which had not been completely resolved from hnRNP-B1 by the C(4) HPLC (see Fig. 2, B and C). The 13-kDa band from the same digest yielded sequence AaRp-s-DGRVv, consistent with that expected for the 13-kDa CNBr fragment of hnRNP-A2/B1. No contaminating protein could be identified from the other automated sequencer calls. At the low level of sample (2 pmol initial yield) the other calls represent reagent and UV background signals.

Analysis of hnRNP-A2/B1 mRNA Expression

Northern analysis (Fig. 5A) demonstrates a wide range of expression of the mRNA for hnRNP-A2/B1 in tumor cell lines, consistent with our radiobinding assays (results not shown). hnRNP-A2/B1 mRNA is also expressed in the single transformed normal bronchial epithelial cell line tested, and in several normal bronchial epithelial cell primary cultures. To quantify the differences in levels of expression, digitized signal intensity of the Northern blot analyzed on a PhosphorImager (Molecular Dynamics) was adjusted for loading differences by quantitation of the 28 S rRNA band photographed under UV light and scanned by laser densitometry (Molecular Dynamics Personal Densitometer). Expression of hnRNP-A2/B1 is highly variable, but in most tumor cell lines is higher than in all six normal lung epithelial cell primary cultures analyzed. By Mann-Whitney rank sum test for comparison of populations with unequal variances, the median value for the tumor cells (873.5, n = 14) is significantly greater than for the normal bronchial epithelial cell cultures (median = 42.0, n = 6) with p < 0.0001. Signal intensity for hnRNP-A2/B1 in the transformed cell line IB3-1 is similar to the tumor cell lines (Fig. 6A). Both NSCLC and SCLC cell lines express hnRNP-A2/B1 mRNA. Northern analysis using a full-length cDNA probe cannot distinguish hnRNP-A2 from -B1, therefore we used reverse transcription-PCR to confirm that both forms of the gene product are expressed. Results show that all tested cell lines and the normal lung expressed both splice forms, and that hnRNP-A2 appears to be the major form in all cases (Fig. 5B).


Figure 5: Expression of hnRNP-A2/B1 mRNA in lung-derived cell cultures. A, Northern analysis of NSCLC cell lines (NCI-H720, H157, HTB58, H520, H676, H1437, A549, H820, H460, and H1155) and SCLC cell lines (NCI-H889, H417, H209, and H345). All cells were harvested in log phase and analyzed as described under ``Experimental Procedures.'' The 28 S rRNA band visualized under UV illumination was used for quantification to carry out the statistical analysis described in the text. For samples run on separate gels, the intensities of the bands was corrected by running NCI-H720 mRNA on each gel as a standard. B, reverse transcription-PCR of mRNA from cell lines NCI-H720, H1355, H157, H1155, normal lung, and normal bronchial epithelium primary culture. Expected size of the products is 280 bp (hnRNP-A2) and 316 bp (hnRNP-B1). reverse transcription-PCR was carried out as described under ``Experimental Procedures.'' Products were analyzed on 2% agarose TBE-gels, transferred to nitrocellulose, and probed with an end-labeled 20-nucleotide primer common to both hnRNP-A2 and -B1.




Figure 6: Proliferation-independent and -dependent control of hnRNP-A2/B1 expression. A, Northern blot hybridization with probe for hnRNP A2/B1 to 10 µg of total RNA from NSCLC (H157, HTB58, and H23), a transformed bronchial epithelium cell line (IB3-1), and normal bronchial epithelium primary cultures (NBE), harvested in log phase (L) or stationary phase (S). B, signal intensity of the Northern blot was determined using a PhosphorImager. The intensities were adjusted for loading different by quantification of the 28 S rRNA band photographed under UV light and scanned by laser densitometry. The corrected signal for the log phase culture of each cell line was set at 100% (log), and the corrected signal for the stationary phase culture (st.) was calculated as a ratio. Data shown are from a single representative experiment.



Biamonti et al.(14) have reported that expression of hnRNP-A1 mRNA, the product of a closely related but distinct gene, is subject to proliferation-dependent regulation in normal fibroblasts and lymphocytes but is proliferation-independent in transformed cell lines and tumors. We analyzed expression of hnRNP-A2/B1 mRNA at different stages of cell growth (Fig. 6). Cells were harvested in either log phase, or stationary phase 4 days after reaching confluence. Our data demonstrate that the levels of the mRNA are proliferation-dependent in normal bronchial epithelial cell primary cultures, but not in transformed cell and tumor cell lines (Fig. 6). In 6/6 normal bronchial epithelial cell primary cultures tested, the levels of hnRNP-A2/B1 mRNA fall after the cells leave log-phase growth (four representative examples from a single experiment are shown in Fig. 6). The level of hnRNP-A2/B1 mRNA does not decline significantly after transformed cells (IB3-1) and tumor cell lines (H157, HTB58, and H23) leave log phase growth (Fig. 6B).


DISCUSSION

The impetus to identify and characterize the protein recognized by 703D4 arose from our clinical experience. We previously described the development of several monoclonal antibodies, including 703D4, which were raised against whole lung tumor cell line extracts(6) . Monoclonal antibody 703D4 was raised to a NSCLC whole cell line immunogen and selected by its ability to discriminate against a SCLC cell line. A cluster analysis demonstrated that 703D4 did not segregate with antibodies which recognize common tumor antigens such as neutral cell adhesion molecules, cytokeratins, or mucins(15) . When 703D4 was used in an immunohistochemical approach in shed bronchial epithelial cells of smoking patients, it correctly detected eventual cancer status with 91% accuracy on average 20 months prior to routine clinical approaches (4) . We have previously mapped the expression of the 703D4 antigen in the epithelium of individuals who had curative lung cancer resections, to evaluate the nature of 703D4 expression in preneoplastic bronchial epithelial cells(16) . We found a complex pattern of expression, but populations of reactive peripheral airway cell frequently stain strongly with 703D4, as well as occasional morphologically normal cells. The antigen detected by 703D4 is therefore a marker for bronchial cells which are committed to a pathway of transformation leading to development of lung cancer. Determination of the identity of the 703D4 antigen was necessary to define its role in the process of carcinogenesis.

We used classical biochemical methods to purify and identify the antigen for 703D4. A series of purification steps including anion exchange chromatography, preparative isoelectric focusing, polymer-based macroporous C(18)-like HPLC and analytical C(4) HPLC resulted in an approximately 25,000-50,000-fold purification of the principal antigen for 703D4. The final C(4) RP-HPLC step of the purification yielded a major 34-kDa protein and a minor 35-kDa protein. The size of fragments generated by CNBr digestion, and internal sequences from both of these molecules, including sequences across and through an alternatively spliced exon, demonstrate that the major and minor antigens are the two gene products of the hnRNP-A2/B1 gene. Recognition that the purified proteins are the authentic 703D4 antigens is apparent from the facts that the immunostaining signal intensities parallel the Coomassie staining intensities (Fig. 2, B and C), and that both purified immunoreactive proteins are mRNA splice variants of a single gene product.

The purified 703D4 antigen was blocked at the amino terminus before SDS-PAGE, as has been previously reported for several hnRNPs(17, 18) . We have not determined the nature of this blocking group but it was removed by CNBr cleavage of the initiator methionine. A variety of post-translational modifications have been reported for members of the hnRNP family, including phosphorylation, methylation of arginine, glycosylation, and ADP-ribosylation(19, 20, 21, 22) . Sequence analysis of four CNBr peptides from hnRNP-A2 and two peptides from hnRNP-B1 revealed no modified amino acid residues which could be identified by Edman degradation and HPLC analysis of the released amino acids. Furthermore, the sizes of the proteins and all CNBr peptides are consistent with that predicted from the position of methionines within the molecule, suggesting no large (>2 kDa) glycosyl or oligonucleotide addition is present. One of our sequences (SS RSG RGG within the 15-kDa CNBr fragment) contains arginines in a similar glycine-rich environment to R of hnRNP-A1, which has been reported to be N^G, N^G-dimethylated in vivo(23, 24) . At the sequencer cycles corresponding to these two arginines there was no apparent loss of signal intensity, suggesting that this site is not methylated to a significant extent in hnRNP-A2 isolated from NCI-H720 lung tumor cell line. Our screening radiobinding analysis of tumor cell lines does not correlate directly with the intensity of signal on Northern analysis. This could imply selectivity of monoclonal antibody 703D4 for absence or presence of a particular post-translational modification. We have commenced a mass spectrometric analysis of protease digests of the purified hnRNP-A2 and hnRNP-B1 to determine whether post-translational modifications are present.

Our data demonstrate highly significant overexpression of hnRNP-A2/B1 in cancer cell lines, and in a transformed bronchial epithelial cell line, compared to normal primary bronchial epithelial cell cultures ( Fig. 5and Fig. 6). We also have preliminary evidence for hnRNP-A2/B1 overexpression in breast tumor cells and transformed breast epithelial cells compared to normal breast epithelial cell primary cultures (data not shown). These findings parallel previous work on the closely related molecule hnRNP-A1, which showed overexpression in several immortalized or transformed cell lines such as epidermal carcinoma cells, promyelocytic cells, SV40-transformed human fibroblasts, and teratocarcinoma cells(14) . Rat neuronal cells also express a high level of hnRNP-A1 mRNA both shortly before and after birth, whereas normal primary fibroblast cultures overexpress hnRNP-A1 only during the logarithmic phase of cell growth(14) . Our data not only demonstrate that hnRNP-A2/B1 is overexpressed in lung epithelial transformed and tumor cells, but also that it is apparently not subject to proliferation-dependent control. These findings of both overexpression and loss of normal transcriptional regulation support our clinical finding that 703D4 detects early tumor cells(4) . The mechanism by which hnRNP-A2/B1 is involved in carcinogenesis and/or tumorigenesis is not clear. Studies on the effect of hnRNP overexpression or knockout on transformation and tumorigenicity are in progress.

Our identification of the 703D4 early lung cancer detection antigen as hnRNP-A2/B1 is provocative in light of the emerging knowledge about the hnRNP group of proteins(25) . The family of hnRNPs have roles in RNA processing, including pre-mRNA exon splicing and splice site choice, and also in transcription, DNA replication, and recombination (19, 26) . hnRNPs are involved in shuttling mRNA from the nucleus to the cytosol, which is consistent with the subcellular fractionation described here and our previously reported immunohistochemical localization(16, 26, 27) . These roles for the hnRNPs indicate these proteins are integral to cellular proliferation. Proliferation markers increase in cells responding normally to injury or during fetal growth and so are not selective for preneoplastic events, that is, they are not specific for cells which have undergone carcinogenesis(28, 29) . However, our clinical findings of increased levels of hnRNP-A2/B1 in exfoliated bronchial cells from patients whose lungs are in the premalignant phases of carcinogenesis suggest a potential causal role for hnRNP-A2/B1 in the process of carcinogenesis(4, 16) . Since hnRNP-A2/B1 have been reported to be major binding protein/s of telomeric sequences, it will be important to evaluate the relationship between telomere regulation and carcinogenesis to evaluate whether hnRNP-A2/B1 is involved in that interaction(30, 31) . Further investigations regarding the ratio of expression of hnRNP-A2 to B1 are warranted in light of previous reports of proliferation dependent changes in the A2/B1 ratio(30, 32) . These data, from several different systems, support a role for hnRNPs in the transformed phenotype, and thereby provide an explanation for our identification of 703D4 as an early lung tumor detection antibody.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Biomarkers and Prevention Research Branch, DCS, NCI, 9610 Medical Center Dr., Rm. 300, Rockville, MD 20850-3300. Tel.: 301-402-3128; Fax: 301-402-4422.

(^1)
The abbreviations used are: NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; hnRNP, heterogeneous nuclear ribonucleoprotein; IEF, isoelectric focusing; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; HFBA, heptafluorobutyric acid; PCR, polymerase chain reaction; MOPS, 3-(N-morpholino)propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; Tricine, N-tris(hydroxymethyl)methylglycine; PVDF, polyvinylidene difluoride.


ACKNOWLEDGEMENTS

We thank Dr. Jill Siegfried (Department of Pharmacology, University of Pittsburgh, Pittsburgh PA) for normal human bronchial epithelial cell cultures; Dr. Pamela Zeitlin (Department of Pediatrics, Johns Hopkins, Baltimore MD) for cell line IB3-1; Dr. Gail Shaw (H. Lee Moffitt Cancer Center, Tampa, FL) for statistical analysis; Dr. Gideon Dreyfuss (HHMI, University of Pennsylvania, Philadelphia, PA) for plasmids containing the cDNAs for hnRNPs and for a monoclonal antibody to hnRNP-A1 (9H10); and Dr. Robert Fisher and Young Kim (Protein Sequencing Laboratory, FCRDC, Frederick MD) for some protein sequence analysis. We also thank Drs. Frank Cuttitta and James Battey for advice in preparing the manuscript.


REFERENCES

  1. Boring, C. C., Squires, T. S., Tong, T., and Montgomery, S. (1994) Ca-Cancer J. Clin. 44, 7-26
  2. Saccomanno, G., Saunders, R. P., and Klein, M. G. (1970) Acta Cytol. 14, 377-381 [Medline] [Order article via Infotrieve]
  3. Frost, J. K., Fontana, R. S., and Melamed, M. R. (1984) Am. Rev. Respir. Dis. 130, 565-570 [Medline] [Order article via Infotrieve]
  4. Tockman, M. S., Gupta, P. K., Myers, J. D., Frost, J. K., Baylin, S. B., Gold, E. B., Chase, A. M., Wilkinson, P. H., and Mulshine, J. L. (1988) J. Clin. Oncol. 6, 1685-1693 [Abstract]
  5. Kyogashima, M., Mulshine, J., Linnoila, R. I., Jensen, S., Magnani, J., Nudelman, E., Hakomori, S., and Ginsburg, V., (1989) Arch. Biochem. Biophys. 275, 309-314 [Medline] [Order article via Infotrieve]
  6. Mulshine, J. L., Cuttitta, F., Bibro, M., Fedorko, J., Fargion, S., Little, C., Carney, D. N., Gazdar, A. F., and Minna, J. D. (1983) J. Immunol. 131, 497-502 [Abstract/Free Full Text]
  7. Neuhoff, V., Arold, N., Taube, D., and Ehrhardt, W. (1988) Electrophoresis 9, 255-262 [Medline] [Order article via Infotrieve]
  8. Krajewski, S., Tanaka, S., Takayama, S., Schibler, M. J., Fenton, W., and Reed, J. (1993) Cancer Res. 53, 4701-4714 [Abstract]
  9. Gross, E., and Morell, J. (1974) Biochem. Biophys. Res. Commun. 59, 1145-1150 [Medline] [Order article via Infotrieve]
  10. Zeitlin, P., Lu, L., Rhim, J., Cutting, G., Stetten, G., Kieffer, K., Craig, R., and Guggino, W. (1991) Am. J. Respir. Cell Mol. Biol. 4, 313-319 [Medline] [Order article via Infotrieve]
  11. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular Biology , pp. 129-156, Elsevier Science Publishing Co., New York
  12. Broder C. C., Earl, P. L., Long, D., Abedon, S. T., Moss, B., and Doms, R. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11699-11703 [Abstract/Free Full Text]
  13. Perton, F. G., Baron, W., Scheffer, A. J., and Beintema, J. J. (1995) Biol. Chem. Hoppe-Seyler 376, 243-247 [Medline] [Order article via Infotrieve]
  14. Biamonti, G., Bassi, M. T., Cartegni, L., Mechta, F., Buvoli, M., Cobianchi, F., and Riva, S. (1993) J. Mol. Biol. 230, 77-89 [CrossRef][Medline] [Order article via Infotrieve]
  15. Beverley, P. C. L., Olabiran, Y., Ledermann, J. A., Bobrow, L. G., and Souhami, R. I. (1991) Br. J. Cancer 63, Suppl. XIV, 10-19
  16. Zhou, J., Jensen, S. M., Steinberg, S. M., Mulshine, J. L., and Linnoila, R. I. (1996) Lung Cancer , in press
  17. Williams, K. R., Stone, K. L., Lopresti, M. B., and Merrill, B. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5666-5670 [Abstract]
  18. Merrill, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H., and Williams, K. R. (1988) J. Biol. Chem. 263, 3307-3313 [Abstract/Free Full Text]
  19. Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993) Annu. Rev. Biochem. 62, 289-321 [CrossRef][Medline] [Order article via Infotrieve]
  20. Soulard, M., Della Valle, V., Siom, M., Pinol-Roma, S., Codogno, P., Bauvy, C., Bellini, M., Lacroix, J. C., Monod, G., and Dreyfuss, G. (1993) Nucleic Acids Res. 21, 4210-4217 [Abstract]
  21. Prasad, S., Walent, J., and Dritschilo, A. (1994) Biochem. Biophys. Res. Commun. 204, 772-779 [CrossRef][Medline] [Order article via Infotrieve]
  22. Bosser, R., Faura, M., Serratosa, J., Renau-Piqueras, J., Pruschy, M., and Bachs, O. (1995) Mol. Cell. Biol. 15, 661-670 [Abstract]
  23. Kumar, A., Williams, K. R., and Szer, W. (1986) J. Biol. Chem. 261, 11266-11273 [Abstract/Free Full Text]
  24. Rajpurohit, R., Lee, S. O., Park, J. O., Paik, W. K., and Kim, S. (1994) J. Biol. Chem. 269, 1075-1082 [Abstract/Free Full Text]
  25. Burd, C. G., and Dreyfuss, G. (1994) Science 29, 615-621
  26. Katz, D., Theodorakis, N. G., Cleveland, D. W., Lindsten, T., and Thompson, C. B. (1994) Nucleic Acids Res. 22, 238-246 [Abstract]
  27. Pinol-Roma, S., and Dreyfuss, G. (1992) Nature 355, 730-732 [CrossRef][Medline] [Order article via Infotrieve]
  28. Risio, M. (1992) J. Cell. Biochem. Suppl. 16G, 79-87
  29. Ganju, R. K., Sunday, M., Tsarwhas, D. G., Card, A., and Shipp, M. A. (1994) J. Clin. Invest. 94, 1784-1791 [Medline] [Order article via Infotrieve]
  30. McKay, S. J., and Cooke, H. (1992) Nucleic Acids Res. 20, 6461-6464 [Abstract]
  31. Ishikawa, F., Matunis, M. J., Dreyfuss, G., and Cech, T. R. (1993) Mol. Cell. Biol. 13, 4301-4310 [Abstract]
  32. Planck, S., Listerud, M., and Buckley, S. (1988) Nucleic Acids Res. 16, 11663-11673 [Abstract]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.