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B1 (hnRNP) in pancreatic tissue from smokers and pancreatic tumor cells
Cancer Letters 183 (2002) 215–220 www.elsevier.com/locate/canlet
Increased expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) in pancreatic tissue from smokers and pancreatic tumor cells Yan Yan-Sanders, George J. Hammons, Beverly D. Lyn-Cook* Division of Molecular Epidemiology, National Center for Toxicological Research, Jefferson, AR 72079, USA Received 10 July 2001; received in revised form 5 March 2002; accepted 13 March 2002
Abstract Pancreatic cancer is a major cause of deaths in the United States, and has one of the lowest 5-year survival rates. Early diagnosis has not been possible due to the lack of reliable early tumor markers. The heterogeneous nuclear ribonucleoprotein A1/B2 (hnRNP) was recently shown to be up-regulated in the early stage of lung cancer. This protein plays an important role in biogenesis and transport of mRNA. Up-regulation of hnRNP usually precedes morphological differentiation and is considered a good biomarker in the early stages of cancer development. Because smoking is a high risk factor for pancreatic cancer, this study examined the expression of hnRNP in human pancreatic tissues from smokers and non-smokers. A two-fold increase in expression of hnRNP was found overall in smokers when compared to non-smokers and smokers who quit (P , 0:05). The increase in expression of hnRNP was higher in female smokers compared to female non-smokers. High levels of expression was also shown in a limited number of human pancreatic adenocarcinomas and two pancreatic tumor cell lines, HPAF-11 and SU 86.86. HP-8, a normal primary pancreatic cell line, did not express hnRNP. These results strongly suggest that up-regulation of hnRNP may be a good candidate for early screening for pancreatic cancer because of its activation in pancreatic tissue from smokers and activation in pancreatic adenocarcinomas. Over-expression of hnRNP has been suggested as evidence that normal transcriptional regulation is altered. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pancreatic cancer; hnRNP gene expression; Smokers
1. Introduction Pancreatic cancer is the fifth leading cause of cancer deaths in the United States, and has one of the lowest 5-year survival rates [1]. Late diagnosis, growth behavior and the biology of the tumor itself are often responsible for such a low survival rate [2]. Males, particularly African Americans, are at a higher risk of developing pancreatic cancer because of diet, genetic, and environmental or life-style factors. * Corresponding author. Tel.: 11-870-543-7965. E-mail address: [email protected] (B.D. Lyn-Cook).
Smoking is one life-style factor that has been associated with a higher risk of developing pancreatic cancer or other pathology of the pancreas. There is a two- to ten-fold increase in risk for pancreatic cancer in smokers [3]. Some chemicals found in cigarettes are known to have adverse physiological and pathological effects on the pancreas [4]. Pancreatic damage from some chemicals found in cigarettes is thought to play a role in the development of pancreatitis, which investigators have recently shown to predispose individuals to developing pancreatic cancer [5]. Early diagnosis of pancreatic cancer is critical for the treatment and increased survival rate for patients with this
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00168-4
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disease. The need for identification of early biomarkers is essential in decreasing the mortality rate of pancreatic cancer. Although some research efforts have focused on identifying early tumor markers, such as serum level tumor-associated antigens, enzymes and/or hormones in the etiology of pancreatic cancer, more research in early detection of pancreatic cancer is needed [6,7]. Up-regulation of the heterogeneous ribonucleoprotein A1/B2 (hnRNP) is thought to play an important role in early stages of carcinogenesis [8]. HnRNP A2/ B1 protein is among the most abundant of the major hnRNP proteins, which are a group of proteins that plays an important role in biogenesis, such as splicing and transport of mRNA in normal cells [9,10]. Increased expression of hnRNP A2/B1 proteins was shown in bronchial epithelial cells by sputum cytology before X-ray diagnosis of lung cancer [11] demonstrating its ability to serve as an early biomarker. Over-expression of hnRNP in transformed lung epithelial and tumor cells when compared to normal epithelial is some evidence that normal transcriptional regulation is altered. HnRNP A2/B1 over-expression has also been detected in other tissues such as transformed breast epithelial and breast tumor cells when compared to normal primary breast epithelial cells [12]. These data suggest that hnRNP family members may be involved in both growth regulation and cancer development [13–15]. Up-regulation of hnRNP precedes morphological differentiation which is another indication that it may be a good biomarker in the early stages of carcinogenesis [16–18]. The present study was conducted to examine the level of expression of hnRNP A2/B1 in human pancreatic tissues from non-smokers (normal) and smokers in order to assess the potential of hnRNP A2/B1 as a early biomarker in a high risk group for pancreatic cancer. This study also examined the level of expression of hnRNP A2/B1 in pancreatic tumor cells and pancreatic tumor tissues.
2. Materials and methods 2.1. Tissue samples Thirty frozen human pancreatic tissues, grouped according to their smoking status and pathological
assessement, were purchased from International Institute for the Advancement of Medicine (Exton, PA) or the University of Alabama at Birmingham Cooperative Human Tissue Network. A research donor medical history form was provided with each sample. A limited number of human pancreatic adenocarcinomas was also examined. All samples were stored at 2808C before total RNA isolation. 2.2. Cell culture of normal and tumor cell lines Pancreatic cancer cell lines (HPAF-11, male and Su 86.86, female) were obtained from American Type Culture Collection (ATCC). HP-8, a normal primary pancreatic cell line, was established in this laboratory. The HPAF-11 and HP-8 cells were grown in MEM medium and Su 86.86 cells were grown in RPMI-1640 medium (GIBCO) without phenol red, supplemented with 10% fetal bovine serum (FBS) and penicillin– streptomycin antibiotic. Cells were grown to 70% confluence, collected, and pelleted. Cell pellets were frozen at 2808C for total RNA isolation. 2.3. RNA preparation and cDNA synthesis Total RNA isolation from cell lines and tissue samples were performed using RNeasy Mini Kits (Qiagen, Valencia, CA). Total RNA was quantified by spectrophotometry at 260 nm. cDNA synthesis was performed with 1 mg of total RNA from each sample group using a cDNA synthesis kit (Clontech Lab, Palo Alto, CA). 2.4. Reverse transcription and quantitative PCR analysis Quantitative reverse transcription-polymerse chain reaction (RT-PCR) was used to determine the expression of hnRNP in human pancreatic tissues and the pancreatic tumor cell lines according to the method reported previously with some modification [17]. The primers designed for hnRNP were F 5 0 -TTAATGGAGGAGGAAGAGGAG-3 0 , and R 5 0 -CTGCATATGCTCTGGTGTCT-3 0 , located at exon 9 and exon 12, which results in a 454 RT-PCR product, according to the gene bank sequence (GeneBank accession no. D28877). b-Actin primers were designed according to the human b-actin sequence (GenBank accession no. M10277): forward primer,
Y. Yan-Sanders et al. / Cancer Letters 183 (2002) 215–220 0
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0
5 -TTGCTATCCAGGCTGTGCTATC-3 ; and the reverse primer, 5 0 -CTTCTCCTTAATGTCACGCACG-3 0 , yielding a 234 bp product. All of the primers were synthesized by Genosys (Woodlands, TX). The PCR 50-ml reaction mixture consisted of cDNA derived from 20 ng of the total RNA, 0.2 mM each of the primers, and 200 mM each of dNTP, 1.25 units of Taq polymerase. PCR was performed for 35 cycles for 50 s at 948C, 40 s at 618C, and 50 s at 728C, and a final extension at 728C for 7 min in the thermocycler. The PCR products were electrophoresed on 2% agarose gels, visualized by ethidium bromide staining, and the bands were quantitated using the Alphaimager 2000 Documentation Analysis System (Alpha Innotech, San Leandro, CA). 3. Results 3.1. Expression of hnRNP in smokers and smokers who quit Overall expression of hnRNP was greater than twofold higher in smokers (0.80 ^ 0.15) when compared to all non-smokers (0.29 ^ 0.158) or smokers who quit (0.17 ^ .01, P , 0:005) (Fig. 1). Examination
Fig. 2. Histogram of the expression level of hnRNP A2/B1 (hnRNP/ actin) in human pancreatic tissue samples grouped by female smokers (FS) and female non-smokers (FNS). The data are presented as the mean ^ standard deviation. *Denotes a significant difference (P , 0:05).
of pancreatic tissues from female smokers and female non-smokers showed that smoking significantly increased the expression of hnRNP (0.80 ^ 0.16 vs. 0.26 ^ 0.09, P , 0:05) (Fig. 2). A sufficient number of pancreatic tissue samples from male non-smokers was unavailable; therefore, statistical differences in comparing this group could not be determined. 3.2. Detection of hnRNP expression in normal and human pancreatic tumor cells HnRNP was expressed at high levels in both pancreatic tumor cell lines, Su 86.86 and HPAF-11 (Fig. 3). Normal pancreatic cells (HP-8) in vitro did not express hnRNP (Fig. 4). In the pancreatic adenocarcinomas tissues examined, different levels of hnRNP were detected (Fig. 5).
4. Discussion Fig. 1. Histogram of the expression levels of hnRNP A2/B1 in all non-smokers (NS), smokers (S), and smokers who quit (SQ). The data are presented as the mean ^ standard deviation. *Denotes a significant difference (P , 0:005).
Recently, studies have suggested overexpression of hnRNP A2/B1, especially of B1, as a useful marker to detect cancer in early stages [19]. Pancreatic cancer is
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Fig. 5. The expression of hnRNP A2/B1 in human pancreatic adenocarcinomas. Individual differences in expression is noted among the different individuals. Fig. 3. The expression of hnRNP A2/B1 in pancreatic tumor cell lines, HPAF-11 and SU 86.86. Lanes 1 and 2 (HPAF-11) and lanes 3 and 4 (SU 86.86).
a disease with a poor prognosis and the etiology is unknown. Therefore, the real challenge for management of this disease is to develop new screening methods or biomarkers that can be used to identify preneoplastic lesions. Members of the hnRNP family play an important role in biogenesis such as transcription, RNA processing (including pre-mRNA exon splicing and splice site choice), and DNA replication and recombination [20,21]. These roles indicate that hnRNP proteins are integral to cell proliferation and
Fig. 4. Histogram of the expression level of hnRNP A2/B1 in pancreatic tumor cell lines, HPAF-11 and SU 86.86, and the normal pancreatic cell line (HP-8).
protein synthesis, thus alterations in expression could affect these two important cellular functions. HnRNP proteins are also known to increase in response to cellular injury, indicating that up-regulation may not be specific only to cells undergoing transformation or carcinogenesis but may be an early indication of cell damage which could ultimately lead to cell transformation [12,22]. In the present study, there was an overall higher level of expression of hnRNP in pancreatic tissue from smokers. When female smokers were compared to female non-smokers, a significantly higher expression of hnRNP was also noted. Samples from male smokers appeared to have a higher level of expression of hnRNP; however, a statistically significant difference could not be demonstrated due to the limited number of non-smoking male samples available (data not shown). This is the first report of an increase in hnRNP expression in smoking tissue from non-cancer pancreatic tissue. Our data are consistent with Zhou et al. [8] who showed higher levels of expression of hnRNP in ‘heavy’ smokers in surrounding lung tissue adjacent to primary lung cancers than in ‘light’ smokers. Our study further demonstrated that smokers who quit had a lower level of expression of hnRNP suggesting some reversal effect of this potential biomarker. This effect could be due to repair of damage when the individual quit smoking because investigators have shown that up-regulation of hnRNP could result from cellular injury [9]. A number of trials with other molecular and morphologic intermediate endpoints have suggested that some of the molecular alterations and morphologic alterations are reversible [23,24]. These studies indicate that hnRNP over-expression in pancreatic tissues from smokers, a high risk group for pancreatic cancer, may make it a good candidate for early screening in pancreatic cancer.
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Aside from cigarette smoking, there are a number of other genetic and metabolic susceptibility factors such as polymorphisms in cytochrome p450 enzymes and the metabolizing capability of glutathone-S-transferase or acetylation that could aid in identifying individuals who are at an increased risk for pancreatic cancer [25]. Although this study showed a significantly higher level of expression in pancreatic tissue from female smokers when compared to non-female smokers, further studies are needed to ascertain whether there is a gender difference in hnRNP exprssion. Both pancreatic tumor cell lines showed expression of hnRNP when compared to a normal primary cell line (HP-8). HPAF-11 cell line is derived from a male and the SU 86.86 cell line was derived from a female. A higher level of expression was noted in the HPAF cell lines. Males have a higher rate of pancreatic cancer than females. High levels of expression of hnRNP in pancreatic tissues from a high risk group such as smokers suggest that this may be a good biomarker for early detection of damage to the pancreas that could ultimately lead to the development of pancreatic cancer. Investigators have suggested up-regulation of hnRNP with actively proliferating cells and that this increase might be a requirement to sustain higher activity of replication, transcription, and translation that occurs during proliferation and/or differentiation [26,27]. This study further demonstrated that smokers who quit smoking showed decreased expression of hnRNP, suggesting reversibility of this biomarker. Further studies are needed to determine the biological significance of overexpression of hnRNP in smoking tissue from pancreas and the role its gene products may play as transcription factors [28] and cell cycle modulators in the early stages of carcinogenesis.
Acknowledgements This research was supported in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration.
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[22] R. Rajpurohit, S.O. Lee, J.O. Park, W.K. Park, S. Kim, Enzymatic methylation of recombinant heterogeneous nuclear RNP protein A1. Dual substrate specificity for S-adnosylmethionine: histone-arginine N-methyltransferase, J. Biol. Chem. 269 (1994) 1075–1082. [23] M.S. Pepe, R. Etzioni, Z. Feng, J.D. Potter, M.L. Thompson, M. Thornquist, M. Winget, Y. Yasui, Phases of biomarker development for early detection of cancer, J. Natl Cancer Inst. 93 (2001) 1054–1061. [24] G.S. Wright, M.E. Gruidl, Early detection and prevention of lung cancer, Curr. Opin. Oncol. 12 (2000) 143–148. [25] G. Liu, P. Ghadirian, D. Vesprini, N. Hamel, A.J. Paradis, G. Lal, S. Gallinger, S.A. Narod, W.D. Foulkes, Polymorphisms in GSTM1, GSTT1 and CYP1A1 and risk of pancreatic adencarcinoma, Br. J. Cancer 82 (2000) 1646–1649. [26] L.P. Montuenga, J. Zhou, I. Avis, M. Vos, A. Martinez, F. Cuttitta, A.M. Treston, M. Sunday, J.L. Mulshine, Expression of heterogeneous nuclear ribonucleoprotein A2/B1 changes with critical stages of mammalian lung development, Am. J. Respir. Cell Mol. Biol. 19 (1998) 554–562. [27] G. Biamonti, M.T. Bassi, L. Cartegni, F. Mechta, M. Buvoli, M. Cobianchi, S. Riva, Human hnRNP protein A1 gene expression: structural and functional characterization of the promoter, J. Mol. Biol. 230 (1993) 77–89. [28] E.F. Michelotti, G.A. Michelotti, A.I. Aronsohn, D. Levens, Heterogeneous nuclear ribonucleoprotein K is a transcription factor, Mol. Cell. Biol. 16 (1996) 2350–2360.
Increased expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) in pancreatic tissue from smokers and pancreatic tumor cells Yan Yan-Sanders, George J. Hammons, Beverly D. Lyn-Cook* Division of Molecular Epidemiology, National Center for Toxicological Research, Jefferson, AR 72079, USA Received 10 July 2001; received in revised form 5 March 2002; accepted 13 March 2002
Abstract Pancreatic cancer is a major cause of deaths in the United States, and has one of the lowest 5-year survival rates. Early diagnosis has not been possible due to the lack of reliable early tumor markers. The heterogeneous nuclear ribonucleoprotein A1/B2 (hnRNP) was recently shown to be up-regulated in the early stage of lung cancer. This protein plays an important role in biogenesis and transport of mRNA. Up-regulation of hnRNP usually precedes morphological differentiation and is considered a good biomarker in the early stages of cancer development. Because smoking is a high risk factor for pancreatic cancer, this study examined the expression of hnRNP in human pancreatic tissues from smokers and non-smokers. A two-fold increase in expression of hnRNP was found overall in smokers when compared to non-smokers and smokers who quit (P , 0:05). The increase in expression of hnRNP was higher in female smokers compared to female non-smokers. High levels of expression was also shown in a limited number of human pancreatic adenocarcinomas and two pancreatic tumor cell lines, HPAF-11 and SU 86.86. HP-8, a normal primary pancreatic cell line, did not express hnRNP. These results strongly suggest that up-regulation of hnRNP may be a good candidate for early screening for pancreatic cancer because of its activation in pancreatic tissue from smokers and activation in pancreatic adenocarcinomas. Over-expression of hnRNP has been suggested as evidence that normal transcriptional regulation is altered. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pancreatic cancer; hnRNP gene expression; Smokers
1. Introduction Pancreatic cancer is the fifth leading cause of cancer deaths in the United States, and has one of the lowest 5-year survival rates [1]. Late diagnosis, growth behavior and the biology of the tumor itself are often responsible for such a low survival rate [2]. Males, particularly African Americans, are at a higher risk of developing pancreatic cancer because of diet, genetic, and environmental or life-style factors. * Corresponding author. Tel.: 11-870-543-7965. E-mail address: [email protected] (B.D. Lyn-Cook).
Smoking is one life-style factor that has been associated with a higher risk of developing pancreatic cancer or other pathology of the pancreas. There is a two- to ten-fold increase in risk for pancreatic cancer in smokers [3]. Some chemicals found in cigarettes are known to have adverse physiological and pathological effects on the pancreas [4]. Pancreatic damage from some chemicals found in cigarettes is thought to play a role in the development of pancreatitis, which investigators have recently shown to predispose individuals to developing pancreatic cancer [5]. Early diagnosis of pancreatic cancer is critical for the treatment and increased survival rate for patients with this
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00168-4
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disease. The need for identification of early biomarkers is essential in decreasing the mortality rate of pancreatic cancer. Although some research efforts have focused on identifying early tumor markers, such as serum level tumor-associated antigens, enzymes and/or hormones in the etiology of pancreatic cancer, more research in early detection of pancreatic cancer is needed [6,7]. Up-regulation of the heterogeneous ribonucleoprotein A1/B2 (hnRNP) is thought to play an important role in early stages of carcinogenesis [8]. HnRNP A2/ B1 protein is among the most abundant of the major hnRNP proteins, which are a group of proteins that plays an important role in biogenesis, such as splicing and transport of mRNA in normal cells [9,10]. Increased expression of hnRNP A2/B1 proteins was shown in bronchial epithelial cells by sputum cytology before X-ray diagnosis of lung cancer [11] demonstrating its ability to serve as an early biomarker. Over-expression of hnRNP in transformed lung epithelial and tumor cells when compared to normal epithelial is some evidence that normal transcriptional regulation is altered. HnRNP A2/B1 over-expression has also been detected in other tissues such as transformed breast epithelial and breast tumor cells when compared to normal primary breast epithelial cells [12]. These data suggest that hnRNP family members may be involved in both growth regulation and cancer development [13–15]. Up-regulation of hnRNP precedes morphological differentiation which is another indication that it may be a good biomarker in the early stages of carcinogenesis [16–18]. The present study was conducted to examine the level of expression of hnRNP A2/B1 in human pancreatic tissues from non-smokers (normal) and smokers in order to assess the potential of hnRNP A2/B1 as a early biomarker in a high risk group for pancreatic cancer. This study also examined the level of expression of hnRNP A2/B1 in pancreatic tumor cells and pancreatic tumor tissues.
2. Materials and methods 2.1. Tissue samples Thirty frozen human pancreatic tissues, grouped according to their smoking status and pathological
assessement, were purchased from International Institute for the Advancement of Medicine (Exton, PA) or the University of Alabama at Birmingham Cooperative Human Tissue Network. A research donor medical history form was provided with each sample. A limited number of human pancreatic adenocarcinomas was also examined. All samples were stored at 2808C before total RNA isolation. 2.2. Cell culture of normal and tumor cell lines Pancreatic cancer cell lines (HPAF-11, male and Su 86.86, female) were obtained from American Type Culture Collection (ATCC). HP-8, a normal primary pancreatic cell line, was established in this laboratory. The HPAF-11 and HP-8 cells were grown in MEM medium and Su 86.86 cells were grown in RPMI-1640 medium (GIBCO) without phenol red, supplemented with 10% fetal bovine serum (FBS) and penicillin– streptomycin antibiotic. Cells were grown to 70% confluence, collected, and pelleted. Cell pellets were frozen at 2808C for total RNA isolation. 2.3. RNA preparation and cDNA synthesis Total RNA isolation from cell lines and tissue samples were performed using RNeasy Mini Kits (Qiagen, Valencia, CA). Total RNA was quantified by spectrophotometry at 260 nm. cDNA synthesis was performed with 1 mg of total RNA from each sample group using a cDNA synthesis kit (Clontech Lab, Palo Alto, CA). 2.4. Reverse transcription and quantitative PCR analysis Quantitative reverse transcription-polymerse chain reaction (RT-PCR) was used to determine the expression of hnRNP in human pancreatic tissues and the pancreatic tumor cell lines according to the method reported previously with some modification [17]. The primers designed for hnRNP were F 5 0 -TTAATGGAGGAGGAAGAGGAG-3 0 , and R 5 0 -CTGCATATGCTCTGGTGTCT-3 0 , located at exon 9 and exon 12, which results in a 454 RT-PCR product, according to the gene bank sequence (GeneBank accession no. D28877). b-Actin primers were designed according to the human b-actin sequence (GenBank accession no. M10277): forward primer,
Y. Yan-Sanders et al. / Cancer Letters 183 (2002) 215–220 0
217
0
5 -TTGCTATCCAGGCTGTGCTATC-3 ; and the reverse primer, 5 0 -CTTCTCCTTAATGTCACGCACG-3 0 , yielding a 234 bp product. All of the primers were synthesized by Genosys (Woodlands, TX). The PCR 50-ml reaction mixture consisted of cDNA derived from 20 ng of the total RNA, 0.2 mM each of the primers, and 200 mM each of dNTP, 1.25 units of Taq polymerase. PCR was performed for 35 cycles for 50 s at 948C, 40 s at 618C, and 50 s at 728C, and a final extension at 728C for 7 min in the thermocycler. The PCR products were electrophoresed on 2% agarose gels, visualized by ethidium bromide staining, and the bands were quantitated using the Alphaimager 2000 Documentation Analysis System (Alpha Innotech, San Leandro, CA). 3. Results 3.1. Expression of hnRNP in smokers and smokers who quit Overall expression of hnRNP was greater than twofold higher in smokers (0.80 ^ 0.15) when compared to all non-smokers (0.29 ^ 0.158) or smokers who quit (0.17 ^ .01, P , 0:005) (Fig. 1). Examination
Fig. 2. Histogram of the expression level of hnRNP A2/B1 (hnRNP/ actin) in human pancreatic tissue samples grouped by female smokers (FS) and female non-smokers (FNS). The data are presented as the mean ^ standard deviation. *Denotes a significant difference (P , 0:05).
of pancreatic tissues from female smokers and female non-smokers showed that smoking significantly increased the expression of hnRNP (0.80 ^ 0.16 vs. 0.26 ^ 0.09, P , 0:05) (Fig. 2). A sufficient number of pancreatic tissue samples from male non-smokers was unavailable; therefore, statistical differences in comparing this group could not be determined. 3.2. Detection of hnRNP expression in normal and human pancreatic tumor cells HnRNP was expressed at high levels in both pancreatic tumor cell lines, Su 86.86 and HPAF-11 (Fig. 3). Normal pancreatic cells (HP-8) in vitro did not express hnRNP (Fig. 4). In the pancreatic adenocarcinomas tissues examined, different levels of hnRNP were detected (Fig. 5).
4. Discussion Fig. 1. Histogram of the expression levels of hnRNP A2/B1 in all non-smokers (NS), smokers (S), and smokers who quit (SQ). The data are presented as the mean ^ standard deviation. *Denotes a significant difference (P , 0:005).
Recently, studies have suggested overexpression of hnRNP A2/B1, especially of B1, as a useful marker to detect cancer in early stages [19]. Pancreatic cancer is
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Fig. 5. The expression of hnRNP A2/B1 in human pancreatic adenocarcinomas. Individual differences in expression is noted among the different individuals. Fig. 3. The expression of hnRNP A2/B1 in pancreatic tumor cell lines, HPAF-11 and SU 86.86. Lanes 1 and 2 (HPAF-11) and lanes 3 and 4 (SU 86.86).
a disease with a poor prognosis and the etiology is unknown. Therefore, the real challenge for management of this disease is to develop new screening methods or biomarkers that can be used to identify preneoplastic lesions. Members of the hnRNP family play an important role in biogenesis such as transcription, RNA processing (including pre-mRNA exon splicing and splice site choice), and DNA replication and recombination [20,21]. These roles indicate that hnRNP proteins are integral to cell proliferation and
Fig. 4. Histogram of the expression level of hnRNP A2/B1 in pancreatic tumor cell lines, HPAF-11 and SU 86.86, and the normal pancreatic cell line (HP-8).
protein synthesis, thus alterations in expression could affect these two important cellular functions. HnRNP proteins are also known to increase in response to cellular injury, indicating that up-regulation may not be specific only to cells undergoing transformation or carcinogenesis but may be an early indication of cell damage which could ultimately lead to cell transformation [12,22]. In the present study, there was an overall higher level of expression of hnRNP in pancreatic tissue from smokers. When female smokers were compared to female non-smokers, a significantly higher expression of hnRNP was also noted. Samples from male smokers appeared to have a higher level of expression of hnRNP; however, a statistically significant difference could not be demonstrated due to the limited number of non-smoking male samples available (data not shown). This is the first report of an increase in hnRNP expression in smoking tissue from non-cancer pancreatic tissue. Our data are consistent with Zhou et al. [8] who showed higher levels of expression of hnRNP in ‘heavy’ smokers in surrounding lung tissue adjacent to primary lung cancers than in ‘light’ smokers. Our study further demonstrated that smokers who quit had a lower level of expression of hnRNP suggesting some reversal effect of this potential biomarker. This effect could be due to repair of damage when the individual quit smoking because investigators have shown that up-regulation of hnRNP could result from cellular injury [9]. A number of trials with other molecular and morphologic intermediate endpoints have suggested that some of the molecular alterations and morphologic alterations are reversible [23,24]. These studies indicate that hnRNP over-expression in pancreatic tissues from smokers, a high risk group for pancreatic cancer, may make it a good candidate for early screening in pancreatic cancer.
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Aside from cigarette smoking, there are a number of other genetic and metabolic susceptibility factors such as polymorphisms in cytochrome p450 enzymes and the metabolizing capability of glutathone-S-transferase or acetylation that could aid in identifying individuals who are at an increased risk for pancreatic cancer [25]. Although this study showed a significantly higher level of expression in pancreatic tissue from female smokers when compared to non-female smokers, further studies are needed to ascertain whether there is a gender difference in hnRNP exprssion. Both pancreatic tumor cell lines showed expression of hnRNP when compared to a normal primary cell line (HP-8). HPAF-11 cell line is derived from a male and the SU 86.86 cell line was derived from a female. A higher level of expression was noted in the HPAF cell lines. Males have a higher rate of pancreatic cancer than females. High levels of expression of hnRNP in pancreatic tissues from a high risk group such as smokers suggest that this may be a good biomarker for early detection of damage to the pancreas that could ultimately lead to the development of pancreatic cancer. Investigators have suggested up-regulation of hnRNP with actively proliferating cells and that this increase might be a requirement to sustain higher activity of replication, transcription, and translation that occurs during proliferation and/or differentiation [26,27]. This study further demonstrated that smokers who quit smoking showed decreased expression of hnRNP, suggesting reversibility of this biomarker. Further studies are needed to determine the biological significance of overexpression of hnRNP in smoking tissue from pancreas and the role its gene products may play as transcription factors [28] and cell cycle modulators in the early stages of carcinogenesis.
Acknowledgements This research was supported in part by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration.
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