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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2008 May 7;134(11):1191–1197. doi: 10.1007/s00432-008-0402-6

Establishment of a new method, transcription–reverse transcription concerted reaction, for detection of plasma hnRNP B1 mRNA, a biomarker of lung cancer

Akemi Sato 1, Naoko Sueoka-Aragane 1,✉, Juichi Saitoh 2, Kazutoshi Komiya 1, Takashi Hisatomi 1, Rika Tomimasu 1, Shinichiro Hayashi 1, Eisaburo Sueoka 1
PMCID: PMC12161729  PMID: 18461365

Abstract

Purpose

Development of an early detection marker is one of the most important strategies for improving overall prognosis in lung cancer patients. We previously reported that hnRNP B1––an RNA binding protein––is overexpressed in lung cancer tissue from the early stage of cancer, and found that hnRNP B1 mRNA is detectable in the plasma of lung cancer patients using real-time RT-PCR. The purpose of this study was to establish a quick and simple method for detecting plasma hnRNP B1mRNA for use in screening for lung cancer.

Methods

TRC, a homogenous method for fluorescence real-time monitoring of isothermal RNA amplification using intercalation activating fluorescence DNA probe, was used to detect plasma hnRNP B1 mRNA.

Results

The detection limit of hnRNP B1 mRNA by TRC using synthetic control RNA or total RNA derived from a lung cancer cell line was 25 or 8.65 × 102 copies, respectively. Using total RNA extracted from 600 μl of plasma, we detected hnRNP B1 mRNA in 39.1% (9/23) of lung cancer patients, with levels ranging from 1.9 to 19,045.5 copies/100 ng RNA, and in 5.2% (5/97) of healthy volunteers. Copy numbers were not associated with age, gender, smoking status, or histological type of cancer. TRC could detect 103 copies of hnRNP B1 mRNA in 10 min.

Conclusion

Detection of plasma hnRNP B1 mRNA by TRC is a quick, easy, and non-invasive method suitable for lung cancer screening.

Keywords: hnRNP B1, Plasma RNA, TRC, Lung cancer

Introduction

Lung cancer is the most common cause of cancer deaths among males in Japan (“Cancer Statistics in Japan” Editorial Board 2005). The overall prognosis is poor and strongly correlated with the stage at diagnosis; 5-year survival is approximately 60% at stage IA, but less than 5% at stage IV (Gazdar and Minna 1999). The cost-effectiveness ratio for treatment of non-small cell lung cancer in the elderly is higher than traditional thresholds (Woodward et al. 2007). In addition, although life expectancy has improved for localized-cancer cases, it has not increased appreciably for distant (metastatic) cases. Considering these facts, early diagnostic tools for lung cancer are urgently needed. We previously reported that hnRNP B1 is overexpressed from the early stage in lung cancer tissue, suggesting that hnRNP B1 has the potential for use in early detection of lung cancers (Sueoka et al. 1999, 2001).

hnRNPs include at least 24 RNA binding proteins ranging in size from 34–120 kDa (Burd and Dreyfuss 1994; Mayeda et al. 1994). The major proteins, such as hnRNP A1, A2, B1, C1 and C2, are nuclear core proteins that bind to nascent RNA polymerase II transcripts and package heterogeneous nuclear RNAs into hnRNP particles (Krecic and Swanson 1999; Barnett et al. 1991). Sequential analysis of hnRNP A2/B1 DNA has revealed that hnRNP B1 is a splicing variant of hnRNP A2 with a mini-exon in the N-terminal encoding 12 amino acids. hnRNP B1 has two tandem repeats of RNA recognition motifs and a glycine-rich domain relevant to protein–protein interaction and RNA–protein interaction (Kozu et al. 1995). Although hnRNP B1 was originally reported to contribute to the splicing of mRNA and its transport from nucleus to cytoplasm (Burd and Dreyfuss 1994; Pinol-Roma and Dreyfuss 1992), other roles in relation to cell proliferation and differentiation, glucose transport, and maintenance of telomere length, all of which might be related to carcinogenesis, have been reported (Hamilton et al. 1999; Ainger et al. 1997; Ishikawa et al. 1993; McKay and Cooke 1992; Camacho-Vanegas et al. 1997; Minoo et al. 1989; Kamma et al. 2001). Recently, we found that hnRNP B1 is involved in the DNA repair system, mediated through the inhibition of DNA-dependent protein kinase activity (Iwanaga et al. 2005).

In 2001, Fleishhacker et al. first reported that hnRNP B1 mRNA was detectable in the plasma of all lung cancer patients examined (Fleischhacker et al. 2001). We also detected hnRNP B1 mRNA in plasma by real-time RT-PCR, and found that hnRNP B1 mRNA was elevated in the plasma of lung cancer patients (Sueoka et al. 2005). Several investigators have suggested that determination of CNAs is useful for detecting and monitoring tumors (Fleischhacker and Schmidt 2007). Because the method is non-invasive, plasma hnRNP B1 mRNA is promising as a potential biomarker for lung cancer screening. However, the concentration of circulating mRNA varies among patients, and more than 1 ml of plasma is necessary to obtain a sufficient amount of total RNA for quantification of the target molecules by RT-PCR in some patients. Because the extraction and purification of RNA following RT-PCR amplification from plasma is costly and time consuming, a simpler and more rapid procedure for detection of the target molecules in plasma RNA is required.

Here, we report the establishment of a new method, TRC, for the detection of hnRNP B1 mRNA using plasma. This method is a completely homogenous method for fluorescence real-time monitoring of isothermal RNA sequence amplification using the INAF DNA probe. TRC has already been applied in quantifying marker genes of Vibrio parahaemolyticus and Mycobacterium bovis BCG starin (Ishiguro et al. 2003; Takakura et al. 2005). TRC is a one-step assay system that comprises both RT and RNA amplification. The possibility of clinical application of this system for lung cancer screening is discussed.

Materials and methods

Collection of plasma samples

Blood was obtained from 97 healthy volunteers who were employees of Saga Medical School, and from 23 lung cancer patients admitted to Saga Medical School Hospital. The volunteers were randomly selected within age decade and gender strata. Samples from the patients were collected before treatment. Blood samples were mixed with one-tenth volume of 3.8% citric acid and subjected to centrifugation at 3,500 rpm at 4°C for 20 min. Supernatants were collected and stored at −80°C until assays were performed. All procedures were performed with informed consent and the study was approved by the Saga University Institutional Review Board.

Preparation of standard RNA for calibration

Standard RNA containing the target region for TRC amplification was prepared by in vitro transcription of SP6 promoter-bearing double-stranded DNA as the template for SP6 RNA polymerase. The methods of DNA template preparation have been described previously (Ishiguro et al. 2003). Briefly, cDNAs were obtained by RT-PCR using primers representing the following coding regions including SP6 promoter sequence: sense 5′-AAG GAT CCG AAT TC A TTT AGG TGA CAC TAT AGA ATA CAA GAA GCG ACT GAG TCC GCG ATG GAG AAA ACT TTA GAA ACT GTT CCT-3′, and antisense 5′-TTT GGA TCC GAA TTC TGG CAA ATA GGA AGA AGC TCA GTA TCG GCT CCT CCC ACC ATA A-3′. The PCR product was digested by EcoRI and purified, followed by subcloning into the pUC19 vector. The resultant RNA was purified by gel filtration with CHROMA SPIN-100 columns (BD Bioscience, Palo Alto, CA). The concentration of the purified RNA was determined by spectrophotometry with absorbance at 260 nm and adjusted to 102–106 copies/5 μl with TE buffer containing 0.25 U/ml of RNase inhibitor and 5 mM dithiothreitol. The RNA was stored at −20°C until use.

Detection of plasma hnRNP B1 mRNA by TRC

Total RNA was isolated from 600 μl of patient’s plasma by ISOGEN-LS® reagent (Nippon gene, Japan) with the modification that plasma was treated with ISOGEN-LS® reagent twice to remove the protein fraction prior to isopropanol precipitation. Calculation of the RNA concentration was based on absorbance at 260 nm. The levels of hnRNP B1 mRNA were determined by TRC, as described elsewhere (Ishiguro et al. 2003) (Fig. 1). Briefly, synthetic oliogonucleotides were used for the TRC reaction, including a pair of amplification primers––the promoter primer 5′-AAT TCT AAT ACG ACT CAC TAT AGG GAG CTG TTC CTT TGG AGA GGA AAA AGA GA-3′ and the reverse primer 5′-CAG GAT CCC TCA TTA CCA CAC A-3′––and a scissors probe 5′-GAA CAG TTT CTA AAG TTT TCT CCA TCG-3′, to initiate the TRC reaction. An INAF probe, 5′-CCA CCA ATA AAG AGC TTA CGG AAC-3′, was prepared for detection of RNA amplification. A volume of 20 μl of the TRC buffer, consisting of a mixture of the substrate solution and the primer solution at a 1:1 (vol/vol) ratio, was added to 5 μl of 20-fold diluted RNA extracts in a thin-wall PCR tube, followed by the addition of 5 μl of the enzyme mix. The mixture was set in a dedicated instrument, TRCRapid-160 (Tosoh Co., Tokyo, Japan), to measure the fluorescence intensity of the reaction mixture incubated at 43°C (excitation wavelength 470 nm, emission wavelength 520 nm). The TRC buffer contained 90 mM Tris–HCl (pH 8.6), 165 mM KCl, 28 mM MgCl2, 1.5 mM dithiothreitol, 0.38 mM dNTP, 3.1 mM NTP, 5.4 mM ITP, 0.3 U/μl RNase inhibitor, 1.5 μM promoter primer, 1.5 μM reverse primer, 0.24 μM scissors probe, 0.23 nM INAF probe, and 16% dimethyl sulfoxide. The enzyme mix consisted of 0.72 mg/ml bovine serum albumin, 12% sorbitol, 1.3 U/μl AMV RTase (Life Technologies) and 28 U/μl T7 RNA polymerase (TaKaRa). The TRCRapid-160 monitor consists of a square incubator block maintained at 43°C and a sliding fluorescence scanning unit, which comprises a light-emitting diode light source to irradiate the excitation light (470 nM) into the tube and a light guide to collect the fluorescence from the bottom of the reaction tube to be introduced into two photomultiplier tubes (520 and 610 nm) at 0.5-min intervals. The reaction time required for fluorescence enhancement to reach a cutoff value of 1.2 was adopted as the detection time for the TRC assay. The cutoff value was chosen to be 3× or more than the standard deviation of the negative control above its fluorescence enhancement at 15 min of the reaction time. Samples for which the detection time was ≤30 min were considered to have a positive signal, and those with positive signals at 520 nm were judged to be positive. The time to reach the cutoff value, 1.2, was used to quantify the initial copy numbers in a sample based on the standard curve constructed with the times of the calibrators.

Fig. 1.

Fig. 1

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Schematic description of the basic steps in TRC. Reverse transcription and RNA amplification are performed isothermally. The reaction is monitored by measuring the fluorescence intensity of the reaction mixture with the INAF probe

Results

hnRNP B1 mRNA detection limit

We first examined the limit of detection with standard hnRNP B1 RNA. Figure 2a shows fluorescence monitoring of the TRC reaction with hnRNP B1 calibrators (standards). The time to detection depended on initial copy number, which ranged from 102 to 106. The time to reach the cutoff value, 1.2, was linearly related to the logarithm of starting copy number (Fig. 2b). The limit of detection was 25 copies (Fig. 2C). Next, hnRNP B1 mRNA isolated from A549 cells, a lung cancer cell line, was determined by TRC. Sequencing analysis of TRC product was performed to confirm the specificity of the amplification. The detection limit for hnRNP B1 mRNA was 10−4 μg, which was estimated to correspond to 8.65 × 102 copies (Fig. 3a). The TRC method detected 105 copies of hnRNP B1 mRNA in 8 min and 103 copies in 10 min, respectively (Fig. 2a).

Fig. 2.

Fig. 2

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a Fluorescence monitoring of the TRC reaction with hnRNP B1 calibrators. Curve labels indicate the starting number of copies of hnRNP B1 calibrator RNA. b Number of starting copies of hnRNP B1 calibrator RNA on a logarithmic scale plotted versus the time required to reach the cutoff level of relative fluorescence intensity, 1.2. Results are for two independent samples. The line is the standard curve for hnRNP B1 calibrator RNA. c Number of starting copies of hnRNP B1 calibrator RNA (less than 100 copies) versus time taken to reach the cutoff level of relative fluorescence intensity, 1.2. Results are for three independent samples

Fig. 3.

Fig. 3

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Fluorescence monitoring of the TRC reaction applied to hnRNP B1 mRNA isolated from A549, a lung cancer cell line. Curve labels indicate the starting amount of total RNA. Ratio of fluorescence intensity is the fluorescence of the samples divided by the background fluorescence

Plasma hnRNP B1 mRNA detected by TRC

In the next step, we adjusted the conditions for RNA isolation from plasma in terms of reagent and volume of plasma. The frequency of hnRNP B1 mRNA detection was higher with ISOGEN-LS® than with ISOGEN® (Table 1). Although the total amounts of RNA were relatively higher when ISOGEN® was used, the purity of RNA was better with ISOGEN-LS® as assessed by the ratio of spectrophotometric absorbance of the samples at 260 nm to that at 280 nm. hnRNP B1 mRNA was more frequently detected, and RNA purity was better, when RNA was isolated from 600 μl of plasma compared with 200 μl (Table 2). Based on these results, we selected ISOGEN-LS® as the RNA isolation reagent and used 600 μl of plasma. With this TRC system, the median concentration of total RNA in the volunteers was 35.2 ng/μl plasma (range 5.4–137.3). hnRNP B1 mRNA was detected in five of the volunteers (5.2%; Table 3). Copy numbers in four of these five volunteers were not high, ranging from 0.1 to 23.3 copies/100 ng RNA, but the other one had a copy number of 364.5 copies/100 ng RNA (data not shown). Positivity was not associated with age, gender, or smoking status. As for the lung cancer patients, the frequency of hnRNP B1 mRNA detection was 39.1% (9/23), ranging from 1.9 to 19,045.5 copies/100 ng RNA (Tables 4 and 5). The median concentration of total RNA was 12.5 ng/μl plasma (range 0.03–96.4), which is similar in magnitude to that of the volunteers. Copy numbers in the patients were not associated with age, gender, smoking status, or histology. The sensitivity and specificity of the TRC method for lung cancer were 39% (9 of 23) and 95% (92 of 97), respectively.

Table 1.

Plasma hnRNP B1 mRNA levels detected by different isolation methods of RNA

No. A260/A280 RNA (ng/μl plasma) hnRNP B1 mRNA
Copies/total RNA Copies/100 ng RNA
Patient 1
 ISOGEN 1.4940 44.28 – –
 ISOGEN-LS 1.5107 11.68 295 1,010.3
Patient 2
 ISOGEN 1.4817 34.18 – –
 ISOGEN-LS 1.6169 9.96 38.1 153.0
Healthy
 ISOGEN 1.4914 32.44 – –
 ISOGEN-LS 1.5467 10.74 – –
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Table 2.

Plasma hnRNP B1 mRNA levels by using different plasma volumes used for RNA isolation

No. A260/A280 RNA (ng/μl plasma) hnRNP B1 mRNA
Copies/total RNA Copies/100 ng RNA
Patient 1
 600 μl 1.5940 22.4 9.79 5.8
 200 μl 1.0971 3.5 – –
Patient 2
 600 μl 1.5934 13.2 8,500 8,559.9
 200 μl 1.7658 1.6 – –
Patient 3
 600 μl 1.6134 1.5 1.45E-09 0.0
 200 μl 1.3116 5.7 – –
Patient 4
 600 μl 1.5764 1.9 217 1,506.9
 200 μl 1.2057 5.2 – –
Patient 5
 600 μl 1.6029 34.0 80.5 31.6
 200 μl 1.5085 32.8 – –
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Table 3.

Detection of plasma hnRNP B1 mRNA in normal, healthy volunteers using TRC

Age Males (%) Females (%) Total (%)
20–29 0 (0/10) 10 (1/10) 5 (1/20)
30–39 20 (2/10) 0 (0/10) 10 (2/20)
40–49 0 (0/10) 10 (1/10) 5 (1/20)
50–59 10 (1/10) 0 (0/10) 5 (1/20)
Above 60 0 (0/12) 0 (0/5) 0 (0/17)
Total 5.8 (3/52) 4.4 (2/45) 5.2 (5/97)
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Table 4.

Detection of plasma hnRNP B1 mRNA levels in lung cancer patients using TRC

No. Age Gender Histology Stage Smoking index TRC (copies/100 ng RNA)
1 70 F Mucoepidermoid IV 0 196.4
2 66 M ad-sq IIIB 800 2.6
3 61 M Small IV 860 401.3
4 76 F ad IIIB 0 23.3
5 72 F ad IA 0 41.7
6 77 M sq IB 300 19,045.5
7 65 M Small IV 1,600 265.1
8 80 M ad IA 1,800 1.9
9 63 M ad IV 240 46.3
10 79 M sq I 750 ND
11 69 M Small IV 1,400 ND
12 77 M sq IIIB 2,000 ND
13 80 M Small IV 1,200 ND
14 72 M ad IA 900 ND
15 60 F ad IA 0 ND
16 60 M Small + LCNEC IIA 940 ND
17 77 M ad IA 400 ND
18 56 M ad IA 255 ND
19 81 F ad IB 0 ND
20 72 F ad IIB 780 ND
21 70 F ad IA 0 ND
22 82 M ad IIIA 1,200 ND
23 74 M sq IA 1,000 ND
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ND not detected, ad-sq adenosquamous cell carcinoma, small small cell carcinoma, ad adenocarcinoma, sq squamous cell carcinoma, LCNEC Large cell neuroendocrine carcinoma

Table 5.

Comparison of plasma hnRNP B1 mRNA levels between lung cancer patients and normal, healthy volunteers

Frequency of detection
Lung cancer patients 9/23 (39.1%)
Healthy volunteers 5/97 (5.2%)
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Discussion

RNA and DNA obtained from plasma or serum are called CNAs. Several investigators have suggested that determination of CNA is useful for detecting and monitoring tumors (Fleischhacker and Schmidt 2007). RNA detected in plasma or serum has been reported to be present as RNA–proteolipid complex (Wieczorek et al. 1985). It is thought that circulating RNA derives from cell secretion rather than cell degradation, because the amount of complex is reduced in cell culture containing cytochalasin B and monensin (Stroun et al. 1987), whereas the complex is not detected in culture media containing polymorphonuclear leukocytes, lymphocytes, or fibroblasts from healthy donors (Wieczorek et al. 1987). These results suggest that determination of CAN is a promising tool for use in cancer screening.

Several candidate markers, such as human tyrosinase, 5T4, mammaglobin, CK-19, Her2/neu, and hnRNP B1 mRNAs, have been investigated using CNAs to evaluate the possibility of cancer detection and monitoring (Kopreski et al. 1999, 2001; Gal et al. 2001; Silva et al. 2001, 2002). At first, most of these investigations were carried out using qualitative RT-PCR. The percentage of positivity in cancer patients ranged by study from 32 to 100%, whereas that in the normal population ranged from 0 to 38%. The wide variation across studies could be due to differences in the markers and assay systems used to detect CNAs. Recently, real-time RT-PCR has been applied to the detection of CNAs to achieve more sensitive and quantitative determination (Lledó et al. 2004; Wong et al. 2004; Miura et al. 2005). Plasma or serum hTERT mRNA was quantified in hepatocellular carcinomas and colorectal cancers (Lledó et al. 2004; Miura et al. 2005). The sensitivity and specificity were 88.2 and 70%, respectively, for hepatocellular carcinoma, and 98 and 64%, respectively, for colorectal cancer, when the cutoff value was defined as the maximum level in the normal population. Plasma β-catenin mRNA levels were associated with cancer progression from normal epithelium to adenoma, and to adenocarcinoma in cases of colon cancer (Wong et al. 2004).

We previously reported that plasma hnRNP B1 mRNA levels assessed using real-time RT-PCR were elevated in lung caner patients (Sueoka et al. 2005). The frequency of hnRNP B1 mRNA elevation in lung cancer cases was 46% whereas that in the normal population was 12%. Positivity was not correlated with staging, and the frequency tended to be high in squamous cell carcinoma, which is compatible with immunohistochemistry findings in lung cancer tissues (Sueoka et al. 1999, 2001). The purpose of the present study was to establish a rapid and simple method for the detection of plasma hnRNP B1 for application to lung cancer screening. TRC is a one-step assay system that comprises both RT and RNA amplification. In addition, this assay system enables us to detect 103 copies of hnRNP B1 mRNA in 10 min. Although the sensitivity was low, the specificity was relatively high, suggesting that, detection of hnRNP B1 mRNA in plasma using TRC is a promising tool for lung cancer screening. To increase the sensitivity, it is necessary to improve the method for isolating RNA from plasma, considering that the frequency of detection of hnRNP B1 mRNA was dependent on the purity of the RNA. Because detection of plasma hnRNP B1 mRNA using TRC is non-invasive, easy, and quick, suitable modification of the TRC method should provide a useful screening system for early detection of lung cancer.

Acknowledgments

This work was supported by grants from the Ministry of Education, Science, Sports and Culture, and the Smoking Research Foundation.

Abbreviations

RT-PCR

Reverse transcription polymerase chain reaction

hnRNP

Heterogeneous nuclear ribonucleoprotein

TRC

Transcription–reverse transcription concerted reaction

INAF

Intercalation activating fluorescence

CNAs

Circulating nucleic acids

TEAA

Triethylammonium acetate

References

  1. Ainger K, Avossa D, Diana AS, Barry C, Barbarese E, Carson JH (1997) Transport and localization elements in myelin basic protein mRNA. J Cell Biol 138:1077–1087 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnett SF, Theiry TA, LeStourgeon WM (1991) The core proteins A2 and B1 exist as (A2)3B1 tetramers in 40S nuclear ribonucleoprotein particles. Mol Cell Biol 11:864–871 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burd CG, Dreyfuss G (1994) Conserved structures and diversity of functions of RNA-binding proteins. Science 265:615–621 [DOI] [PubMed] [Google Scholar]
  4. Camacho-Vanegas O, Weighardt F, Ghigna C, Amaldi F, Riva S, Biamonti G (1997) Growth-dependent and growth-independent translation of messengers for heterogeneous nuclear ribonucleoproteins. Nucleic Acids Res 25:3950–3954 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. “Cancer Statistics in Japan” Editorial Board (2005) Number of deaths and proportional mortality rates from malignant neoplasms by site in Japan. In: Nomura K, Sobue T, Nakatani H et al (eds) Cancer statistics in Japan-2005. Tokyo Foundation for Promotion of Cancer Research, pp 36–39
  6. Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer––a survey. Biochim Biophys Acta 1775:181–232 [DOI] [PubMed] [Google Scholar]
  7. Fleischhacker M, Beinert T, Ermitsch M, Seferi D, Possinger K, Engelmann C, Jandrig B (2001) Detection of amplifiable messenger RNA in the serum of patients with lung cancer. Ann N Y Acad Sci 945:179–188 [DOI] [PubMed] [Google Scholar]
  8. Gal S, Fidler C, Lo YM, Chin K, Moore J, Harris AL, Wainscoat JS (2001) Detection of mammaglobin mRNA in the plasma of breast cancer patients. Ann N Y Acad Sci 945:192–194 [DOI] [PubMed] [Google Scholar]
  9. Gazdar AF, Minna J (1999) Molecular detection of early lung cancer. J Natl Cancer Inst 91:299–301 [DOI] [PubMed] [Google Scholar]
  10. Hamilton BJ, Nichols RC, Tsukamoto H, Boado RJ, Pardridge WM, Rigby WFC (1999) hnRNP A2 and hnRNP L bind the 3′UTR of glucose transporter 1 mRNA and exist as a complex in vivo. Biochem Biophys Res Commun 261:646–651 [DOI] [PubMed] [Google Scholar]
  11. Ishiguro T, Saitoh J, Horie R, Hayashi T, Ishizuka T, Tsuchiya S, Yasukawa K, Kido T, Nakaguchi Y, Nishibuchi M, Ueda K (2003) Intercalation activating fluorescence DNA probe and its application to homogeneous quantification of a target sequence by isothermal sequence amplification in a closed vessel. Anal Biochem 314:77–86 [DOI] [PubMed] [Google Scholar]
  12. Ishikawa F, Matunis MJ, Dreyfuss G, Cech TR (1993) Nuclear proteins that bind the pre-mRNA 3′ splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n. Mol Cell Biol 13:4301–4310 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Iwanaga K, Sueoka N, Sato A, Hayashi S, Sueoka E (2005) Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity. Biochem Biophys Res Commun 333:888–895 [DOI] [PubMed] [Google Scholar]
  14. Kamma H, Fujimoto M, Fujiwara M, Matsui M, Horiguchi H, Hamasaki M, Satoh H (2001) Interaction of hnRNP A2/B1 isoforms with telomeric ssDNA and the in vitro function. Biochem Biophys Res Commun 280:625–630 [DOI] [PubMed] [Google Scholar]
  15. Kopreski MS, Benko FA, Kwak LW, Gocke CD (1999) Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 5:1961–1965 [PubMed] [Google Scholar]
  16. Kopreski MS, Benko FA, Gocke CD (2001) Circulating RNA as a tumor marker: detection of 5T4 mRNA in breast and lung cancer patient serum. Ann N Y Acad Sci 945:172–178 [PubMed] [Google Scholar]
  17. Kozu T, Henrich B, Schafer KP (1995) Structure and expression of the gene (HNRPA2B1) encoding the human hnRNP protein A2/B1. Genomics 25:365–371 [DOI] [PubMed] [Google Scholar]
  18. Krecic AM, Swanson MS (1999) hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol 11:363–371 [DOI] [PubMed] [Google Scholar]
  19. Lledó SM, Garcia-Granero E, Dasí F, Ripoli R, García SA, Cervantes A, Aliño SF (2004) Real time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA in patients with colorectal cancer. Colorectal Dis 6:236–242 [DOI] [PubMed] [Google Scholar]
  20. Mayeda A, Munroe SH, Caceres JF, Krainer AR (1994) Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J 13:5483–5495 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McKay SJ, Cooke H (1992) hnRNP A2/B1 binds specifically to single stranded vertebrate telomeric repeat TTAGGGn. Nucleic Acids Res 20:6461–6465 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Minoo P, Sullivan W, Solomon LR, Martin TE, Toft DO, Scott RE (1989) Loss of proliferative potential during terminal differentiation coincides with the decreased abundance of a subset of heterogeneous ribonuclear proteins. J Cell Biol 109:1937–1946 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miura N, Maeda Y, Kanbe T, Yazama H, Takeda Y, Sato R, Tsukamoto T, Sato E, Marumoto A, Harada T, Sano A, Kishimoto Y, Hirooka Y, Murawaki Y, Hasegawa J, Shiota G (2005) Serum human telomerase reverse transcriptase messenger RNA as a novel tumor marker for hepatocellular carcinoma. Clin Cancer Res 11:3205–3209 [DOI] [PubMed] [Google Scholar]
  24. Pinol-Roma S, Dreyfuss G (1992) Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature 355:730–732 [DOI] [PubMed] [Google Scholar]
  25. Silva JM, Dominguez G, Silva J, Garcia JM, Sanchez A, Rodriguez O, Provencio M, España P, Bonilla F (2001) Detection of epithelial messenger RNA in the plasma of breast cancer patients is associated with poor prognosis tumor characteristics. Clin Cancer Res 7:2821–2825 [PubMed] [Google Scholar]
  26. Silva JM, Rodriguez R, Garcia JM, Muñoz C, Silva J, Dominguez G, Provencio M, España P, Bonilla F (2002) Detection of epithelial tumour RNA in the plasma of colon cancer patients is associated with advanced stages and circulating tumour cells. Gut 50:530–534 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Stroun M, Anker P, Beljanski M, Henri J, Lederrey C, Ojha M, Maurice PA (1987) Presence of RNA in the nucleoprotein complex spontaneously released by human lymphocytes and frog auricles in culture. Cancer Res 38:3546–3554 [PubMed] [Google Scholar]
  28. Sueoka E, Goto Y, Sueoka N, Kai Y, Kozu T, Fujiki H (1999) Heterogeneous nuclear ribonucleoprotein B1 as a new marker of early detection for human lung cancers. Cancer Res 59:1404–1407 [PubMed] [Google Scholar]
  29. Sueoka E, Sueoka N, Goto Y, Matsuyama S, Nishimura H, Sato M, Fujimura S, Chiba H, Fujiki H (2001) Heterogeneous nuclear ribonucleoprotein B1 as early cancer biomarker for occult cancer of human lungs and bronchial dysplasia. Cancer Res 61:1896–1902 [PubMed] [Google Scholar]
  30. Sueoka E, Sueoka N, Iwanaga K, Sato A, Suga K, Hayashi S, Nagasawa K, Nakachi K (2005) Detection of plasma hnRNP B1 mRNA, a new cancer biomarker, in lung cancer patients by quantitative real-time polymerase chain reaction. Lung Cancer 48:77–83 [DOI] [PubMed] [Google Scholar]
  31. Takakura S, Tsuchiya S, Isawa Y, Yasukawa K, Hayashi T, Tomita M, Suzuki K, Hasegawa T, Tagami T, Kurashima A, Ichiyama S (2005) Rapid detection of Mycobacterium tuberculosis in respiratory samples by transcription–reverse transcription concerted reaction with an automated system. J Clin Microbiol 43:5435–5439 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wieczorek AJ, Rhyner C, Block LH (1985) Isolation and characterization of an RNA–proteolipid complex associated with the malignant state in humans. Proc Natl Acad Sci USA 82:3455–3459 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wieczorek AJ, Sitaramam V, Machleidt W, Rhyner K, Perruchoud AP, Block LH (1987) Diagnostic and prognostic value of RNA–proteolipid in sera of patients with malignant disorders following therapy: first clinical evaluation of a novel tumor marker. Cancer Res 47:6407–6412 [PubMed] [Google Scholar]
  34. Wong SC, Lo SF, Cheung MT, Ng KO, Tse CW, Lai BS, Lee KC, Lo YM (2004) Quantification of plasma beta-catenin mRNA in colorectal cancer and adenoma patients. Clin Cancer Res 10:1613–1617 [DOI] [PubMed] [Google Scholar]
  35. Woodward RM, Brown ML, Stewart ST, Cronin KA, Cutler DM (2007) The value of medical interventions for lung cancer in the elderly: results from SEER-CMHSF. Cancer 110:2511–2518 [DOI] [PubMed] [Google Scholar]

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