(Received for publication, November 13, 1995; and in revised form, February 17, 1996)
From the
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-like, and analytical C
reverse
phase high performance liquid chromatography. After
25-50,000-fold purification, the principal immunostaining protein
was >95% pure by Coomassie staining. The NH
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
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.
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 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) ()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.
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 g for 20 min. The supernatant was then diluted 3-fold with
deionized water and adjusted to pH 6.5 for ion-exchange chromatography.
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.
As the last stage in the purification, the positive
fractions were applied to a 2.1 mm 25 cm Vydac analytical
C
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.
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 10
cpm/ml of probe was used for
each Northern analysis.
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 was identifiable (M
= 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-reversed phase HPLC
purification of 703D4 antigen. 703D4 immunoreactive fractions from
methanol/HFBA were injected onto a 2.1 mm
25-cm Vydac C
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 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.
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 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
column, and eluted with a slow gradient of acetonitrile
(0.2%/min). Immunoblotting analysis of the C
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-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.
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-terminal sequences of
peptides isolated from CNBr digest of purified 703D4 antigen. The
NH
-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 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
-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
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.
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).
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-like HPLC and
analytical C
HPLC resulted in an approximately
25,000-50,000-fold purification of the principal antigen for
703D4. The final C
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
, N
-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.