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Japanese Journal of Clinical Oncology Pages 723-728

Expression of [alpha]-Fetoprotein and Prostate-specific Antigen Genes in Several Tissues and Detection of mRNAs in Normal Circulating Blood by Reverse Transcriptase-Polymerase Chain Reaction
Introduction
Materials And Methods
   Probes
   Northern Blotting
   RNA Preparation and RT-PCR
Results
   Tissue Specificity of AFP and PSA Gene Expression
   Detection of AFP and PSA mRNA by RT-PCR in Cancers and Normal Peripheral Blood
Discussion
Acknowledgments
References
Expression of [alpha]-Fetoprotein and Prostate-specific Antigen Genes in Several Tissues and Detection of mRNAs in Normal Circulating Blood by Reverse Transcriptase-Polymerase Chain Reaction

Expression of [alpha]-Fetoprotein and Prostate-specific Antigen Genes in Several Tissues and Detection of mRNAs in Normal Circulating Blood by Reverse Transcriptase-Polymerase Chain Reaction

Tomoyoshi Ishikawa1, Hironobu Kashiwagi1, Yoko Iwakami1, Misako Hirai1, Tomonori Kawamura1, Yuji Aiyoshi2, Toru Yashiro2, Yoshihiro Ami3, Kazuhiko Uchida1 and Masanao Miwa1

1Department of Biochemistry and Molecular Oncology, Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, 2Department of Surgery, Institute of Clinical Medicine, University of Tsukuba and 3Department of Urology, Tsukuba Gakuen Hospital, Tsukuba, Ibaraki, Japan

Background: [alpha]-Fetoprotein (AFP) and prostate-specific antigen (PSA) in serum are widely used as tumor markers in the evaluation of prognosis and management of patients with hepatocellular carcinoma and prostate cancer, respectively. To establish the molecular diagnosis of cancer, reverse transcriptase polymerase chain reaction (RT-PCR) for AFP and PSA was used to identify circulating cancer cells in the blood of cancer patients. Here, we examined the tissue-specificity of AFP and PSA and tested whether AFP and PSA are suitable targets in the detection of certain cancer cells by RT-PCR using peripheral blood samples.
Methods: Tissue specificity of AFP and PSA was analyzed by Northern blotting and RT-PCR. Probes for AFP and PSA were hybridized with poly A+ RNAs from 50 human tissues. RT-PCR for AFP and PSA mRNA was performed using several cancerous tissues and normal tissues and peripheral blood cells from seven healthy volunteers.
Results: Broad expression of AFP was observed in several tissues and a large amount of AFP mRNA was found in fetal liver. PSA was expressed in prostate, salivary gland, pancreas and uterus. By RT-PCR, AFP and PSA mRNA were detected in several tumors, including salivary pleomorphic adenoma, hilar bile duct carcinoma, pancreatic carcinoma, transitional cell carcinoma of urinary bladder and thyroid papillary carcinoma. Furthermore, AFP and PSA mRNAs were frequently detected by RT-PCR, even in peripheral blood cells from healthy volunteers.
Conclusions: Neither AFP nor PSA showed tissue-specific expression. AFP and PSA mRNA were detected in several diseased and non-diseased tissues and normal circulating blood by RT-PCR.

Key words: tissue-specific expression - [alpha]-fetoprotein - prostate-specific antigen - reverse transcriptase polymerase chain reaction - molecular diagnosis

INTRODUCTION

In Japan, about 300 000 people die yearly of cancer, making this disease the leading cause of death in adults (1). Non-invasive and highly sensitive diagnostic methods against cancer will undoubtedly have a major impact on cancer diagnosis and therapy. [alpha]-Fetoprotein (AFP) and prostate-specific antigen (PSA) in serum are valuable biological markers for the diagnosis, prognosis and management of patients with hepatocellular carcinoma (HCC) and prostate cancer, respectively (2,3). AFP is a serum protein mainly synthesized in mammalian fetal liver cells (4-8). PSA is a glycoprotein (9,10) up-regulated by androgenic hormones at the transcriptional level (11) and mainly produced in the prostate. Recently, the detection of circulating cancer cells by reverse transcriptase-polymerase chain reaction (RT-PCR) for the evaluation of post-operative prognosis, molecular staging of cancers (12) and diagnosis of micro-metastasis (13) has been reported. In these reports, tumor-specific mRNA has been employed to detect circulating cancer cells in peripheral blood cells of cancer patients. Detection of AFP and PSA mRNA by RT-PCR in peripheral blood has become one of the most useful molecular biomarkers (12,14-20) in cancer diagnosis. PCR is an extremely sensitive method to detect even small amounts of DNA/RNA sequences. Because of its high sensitivity, false-positive results can possibly occur even if AFP and PSA mRNA are used as targets for RT-PCR. Recent studies have reported detection of PSA mRNA by RT-PCR in several cancers other than prostate cancer (21-25). Expression of AFP in normal renal cells has also been detected (26). Low levels of PSA and AFP transcripts in peripheral blood from non-cancer patients can possibly affect the ability of RT-PCR to detect circulating cancer cells. Tissue specificity of gene expression of PSA and AFP is crucial in the establishment of cancer diagnosis by RT-PCR using peripheral blood.

In this study, we analyzed the tissue specificity of AFP and PSA in 43 human adult and seven fetal tissues by Northern blotting and examined the expression of AFP and PSA by RT-PCR using several tumors and normal peripheral blood cells.

MATERIALS AND METHODS

Probes

cDNA probes for AFP and PSA mRNAs were prepared by RT-PCR using total RNA from normal liver and prostate, respectively. The sequences of AFP primers were 5-GTT GCC AAC TCA GTG AGG AC-3 for the forward primer (AFP-F) and 5-GAG CTT GGC ACA GAT CCT TA-3 for the reverse primer (AFP-R). The sequences of PSA primers were 5-CCC ACA CCC GCT CTA CGA TA-3 for the forward primer (PSA-F) and 5-ACC TTC TGA GGG TGA ACT TGC G-3 for the reverse primer (PSA-R). The PCR products of AFP and PSA cDNAs are 240 and 289 base pairs in length, respectively. The RT-PCR products of AFP and PSA were purified with a MERmaid Kit (BIO 101, Vista, CA) and subcloned into pCR 2.1 (Original TA Cloning Kit, Invitrogen, San Diego, CA). The resulting plasmids were digested by EcoRI and the inserts were electrophoretically separated on a 0.8% agarose gel for purification. Human ubiquitin cDNA (CLONTECH, Palo Alto, CA) was used for standardization. The isolated DNA fragments were radiolabeled with [[alpha]-32P]dCTP using a Multiprime DNA Labeling System (Amersham, UK). Plasmid inserts were sequenced by the dye primer method using a DNA Sequencing Kit with Dye Primer Cycle Sequencing Ready Reaction (Perkin-Elmer-Cetus, Foster City, CA) with an ABI PRISM 310 Genetic Analyzer (Perkin-Elmer-Cetus).

Northern Blotting

Tissue specificity was analyzed by using a Human RNA Master Blotting (CLONTECH). Hybridization was performed according to the manufacturer's protocol. Amounts of 20 ng of probes were labeled with [[alpha]-32P]dCTP and then added to a mixture of 25 µg of human Cot-1 DNA (GIBCO BRL, Rockville, MD), 125 µg of sheared salmon testis DNA and 5 ml of ExpressHyb solution (CLONTECH). The blot was incubated at 65°C for 16 h. After washing four times with 2× SSC and 1% SDS at 65°C for 20 min. two additional 20 min washes were performed in 0.1× SSC and 0.5% SDS at 65°C. The damp blot was exposed on an Imaging Plate (FujiPhoto Film, Tokyo). Dots on the autoradiograph were analyzed and quantified with a BAS 5000 Imaging Analyzer (FujiPhoto Film). To determine the tissue specificity of the gene expression, the intensity percentage (% intensity) of each dot was calculated as follows according to the manufacturer's protocol. The raw data of the position of yeast transfer RNA on the blot (H2) was set as the background of the blot (data not shown in Figs 1 and 2). Signal intensity values for each dot were divided by the product of the original mRNA amounts (ng) and the scan area (mm2). The value thus obtained in certain tissues was divided by the sum of all obtained values of the blotting and shown as % intensity. The ratio of the expressed mRNA in each tissue was obtained from this calculation.


Figure 1. Expression of AFP and PSA genes in various tissues. AFP and PSA cDNAs were hybridized with poly A+ RNAs from 50 tissues. Hybridization of ubiquitin cDNA was performed for standardization. AFP was expressed in more than 30 types of tissue. A strong signal was observed in fetal liver. PSA was expressed mainly in prostate, salivary gland and pancreas. The type and position of poly A+ RNAs on the blot are as follows: A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hipocampus; A8, medulla oblongata; B1, occipital lobe; B2, putamen; B3, substantia nigra; B4, temporal lobe; B5, thalamus; B6, subthalamic nucleus; B7, spinal cord; C1, heart; C2, aorta; C3, skeletal muscle; C4, colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; D1, testis; D2, ovary; D3, pancreas; D4, pituitary gland; D5, adrenal gland; D6, thyroid gland; D7, salivary gland; D8, mammary gland; E1, kidney; E2, liver; E3, small intestine; E4, spleen; E5, thymus; E6, peripheral lymphocytes; E7, lymph node; E8, bone marrow; F1, appendix; F2, lung; F3, trachea; F4, placenta; G1, fetal brain; G2, fetal heart; G3, fetal kidney; G4, fetal liver; G5, fetal spleen; G6, fetal thymus; and G7, fetal lung.


Figure 2. Intensity percentage of AFP and PSA gene expression. Each bolt was analyzed using Phosphorimager and quantified with the BAS 5000 Imaging Analyzer. The % intensity for each tissue was calculated as described in Materials and Methods. Tissue samples are indicated in groups from A to G. From the left, A1-8, B1-7, C1-8, D1-8, E1-8, F1-4 and G1-7 are shown. Definitions as in Fig. 1.

RNA Preparation and RT-PCR

Total RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform extraction (AGPC) method (27) from the following tissues: normal liver, hilar bile duct carcinoma, prostate cancer, benign prostatic hypertrophy, normal prostate, pancreatic carcinoma, normal pancreas, pleomorphic adenoma of parotid gland, normal parotid gland, bladder cancer, thyroid papillary carcinoma and normal thyroid gland. Whole blood was subjected to a Ficcoll-Conray gradient (IBL, Fujioka) and the nucleate cells were collected. Total RNAs from 5 ml of peripheral whole blood of seven healthy volunteers were also extracted by the AGPC method. Total RNA was finally dissolved in diethylpyrocarbonate-treated water to a final concentration of 1 µg/µl. Prior to reverse transcription, the total RNA solution was heated at 70°C for 10 min and then immediately placed on ice for 5 min. cDNA was synthesized using a RNA PCR Core Kit (Perkin-Elmer-Cetus) from 1 µg of total RNA in 20 µl of reaction mixture containing 1× PCR Buffer II, 1 mM dNTP, 2.5 mM oligo-d(T)16, 20 units of RNasin and 50 units of murine leukemia virus (MuLV) reverse transcriptase. Reverse transcription was carried out at 42°C for 15 min. A 5 µl volume of the reaction mixture was used for PCR. PCR was carried out in 50 µl of reaction mixture containing 1× PCR Buffer II containing AmpliTaq Gold (Perkin-Elmer-Cetus) DNA polymerase. To obtain the most effective amplification by AmpliTaq Gold DNA polymerase, the PCR cycle was increased up to 50 cycles according to the manufacturer's protocol. PCR cycles for AFP and PSA were as follows: initial denaturation at 95°C for 10 min, followed by 50 cycles of 94°C for 1 min, 58°C for 2 min, 72°C for 3 min and final extension at 72°C for 7 min. RT-PCR of [beta]-actin was used as an internal control as described (28). The sequences of [beta]-actin primers are 5-AGA GAT GGC CAC GGC TGC TT-3 for the forward primer in exon 4 and 5-ATT TGC GGT GGA CGA TGG AG-3 for the reverse primer in exon 6. The PCR product is 406 bp in length. After reverse transcription, amplification was performed with 0.2 U of AmpliTaq DNA polymerase (Perkin-Elmer-Cetus) in 2.5 mM MgCl2, 10 mM Tris (pH 8.3), 50 mM KCl, 0.01% gelatin (w/v), 0.25 mM of each dNTP and 5 pmol of each primer, by a precycle at 94°C for 3 min and subsequently 40 cycles of 1 min at 94°C, 1 min at 60°C and 2 min at 72°C in a thermal cycler (Perkin-Elmer-Cetus). RT-PCR products of AFP, PSA and [beta]-actin were separated electrophoretically on a 2.0% agarose gel and detected with ethidium bromide staining.

RESULTS

Tissue Specificity of AFP and PSA Gene Expression

Fig. 1 shows the expression of AFP and PSA genes in 50 human tissues including fetal organs. The total quantity of mRNA in each dot ranges between 95 (E6; peripheral leukocytes) and 461 ng/dot (D3; pancreas). The AFP gene was highly expressed in fetal liver. However, the broad expression pattern of the AFP gene was observed. Positive signals were found in several kinds of tissue, including peripheral leukocytes. AFP mRNA was also detected in various kinds of fetal tissues. Fig. 2 shows the % intensity of expression of AFP and PSA genes to evaluate the tissue specificity of the gene expression. A high % intensity was observed for AFP mRNA in fetal liver (49.3%), fetal spleen (4.2%), bladder (2.1%), frontal lobe (1.9%) and thalamus (1.9%). Peripheral leukocytes showed expression of AFP at 1.64% intensity. Thus, AFP mRNA was detected in almost all tissues, including peripheral leukocytes, even at low levels.

The PSA gene also showed broad expression patterns as shown in Figs 1 and 2. Strong signals were observed in prostate, salivary gland, pancreas and uterus. The % intensities of PSA were prostate 82.5%, salivary gland 2.9%, pancreas 1.7% and uterus 1.0%. Signals for PSA were detected in almost all tissues, including peripheral leukocytes (0.4%). Small amounts of PSA mRNA were also found in various kinds of adult tissues. Fetal kidney was the only tissue where the signal for PSA mRNA was not detected.

Detection of AFP and PSA mRNA by RT-PCR in Cancers and Normal Peripheral Blood

We analyzed AFP and PSA expression in several cancer tissues to examine the cancer specificity of their expression. AFP mRNA was detected in normal liver, hilar bile duct carcinoma, prostate cancer, benign prostatic hypertrophy, normal prostate, pancreatic carcinoma, normal pancreas, pleomorphic adenoma of parotid gland, normal parotid gland, bladder cancer, thyroid papillary carcinoma and normal thyroid gland (Fig. 3). PSA mRNA was detected not only in prostate but also in normal liver, hilar bile duct carcinoma, pancreas carcinoma, normal pancreas, pleomorphic adenoma of parotid gland, normal parotid gland, bladder cancer, thyroid papillary carcinoma and normal thyroid gland (Fig. 3). By RT-PCR, AFP mRNA was detected in peripheral blood from six out of seven healthy volunteers and PSA mRNA was also detected in five out of seven healthy volunteers (Fig. 4). [beta]-actin mRNA as internal control was detected in all cases by RT-PCR. In some cases, contaminated genomic DNA was observed at about 432 bp in PCR for PSA, whereas no band from genomic DNA was observed in PCR for AFP because of the large size ([sim]1600 bp) of the expected PCR product from genomic DNA and the small amount of contaminated genomic DNA.


Figure 3. Detection of AFP and PSA mRNA in various cancers by RT-PCR. AFP and PSA transcripts were 240 and 289 bp, respectively. Lanes in each panel are as follows: 1, normal liver; 2, hilar bile duct carcinoma; 3, prostatic cancer; 4, benign prostatic hypertrophy; 5, normal prostate; 6, pancreatic carcinoma; 7, normal pancreas; 8, pleomorphic adenoma of parotid gland; 9, normal parotid gland; 10, bladder cancer; 11, thyroid papillary carcinoma; 12, normal thyroid gland; 13, DNA from normal lymphocytes as a negative control; NTC, no template control; M, DNA marker (pBR322 HinfI digest).


Figure 4. Detection of AFP and PSA mRNA in peripheral blood cells from healthy volunteers by RT-PCR. Lanes 1-7, peripheral blood cells from healthy volunteers; NTC, no template control; M, DNA marker (pBR322 HinfI digest).

DISCUSSION

We have found that various human tissues, including peripheral leukocytes, expressed the AFP and PSA genes. mRNAs for these genes were frequently detected in normal circulating blood. Northern blot analysis using poly A+ RNAs from 50 human tissues including fetal organs revealed that both AFP and PSA expression were not tissue specific. Various tissues, including peripheral blood, showed positive spots on the blot. Furthermore, RT-PCR for AFP and PSA mRNAs indicated the presence of these mRNAs in normal peripheral blood cells. These data suggest a limitation of PCR-based methods with these weak tumor- or tissue-specific mRNAs as targets. Because of the very high sensitivity of PCR, low levels of these tumor-specific transcripts, as shown by Northern blotting (Figs 1 and 2), can be detected in peripheral blood cells from non-cancer patients and healthy volunteers.

By quantification of the signals on the Northern blot, several tissues expressed AFP at about 1/25th of the level of the fetal liver. By RT-PCR, we found AFP mRNA in cancers and benign tumors such as adenoma of parotid gland, bile duct cancer, pancreas cancer, bladder cancer and thyroid cancer. AFP gene expression in neonatal rat kidney has been reported (26). We also showed AFP expression in fetal kidney.

PSA is believed to be expressed exclusively in prostatic epithelial cells. However, in the present study, expression of the PSA gene was observed not only in the prostate but also in various other tissues including pancreas, salivary gland and uterus as well as peripheral blood. However, the expression levels in these tissues, excluding prostate, were very low. The % intensity for peripheral leukocytes was 0.4%, whereas prostate showed 82.5% intensity. Even at these low expression levels, we detected PSA mRNA in normal tissues such as liver, pancreas, parotid gland and thyroid gland and also in several cancers by RT-PCR (Figs 3 and 4). PSA mRNA was frequently detected in peripheral blood cells from healthy volunteers by RT-PCR. These data coincide with previous reports indicating expression of PSA in non-prostate cells including normal blood cells (29-32). Convincing evidence has been described for expression of PSA in normal tissues such as salivary gland (31), lung (33) and endometrium (34) and also in tumors such as lung cancer (23,35), breast cancer (21), ovarian tumor (24) and other tumors (25). Recent RT-PCR studies suggested expression of the PSA gene in breast and lung cancers (21-23,35).

The present study clearly showed the broad expression pattern of the AFP and PSA genes and no specificity for certain cancers. Detection of the circulating cancer cells by RT-PCR for cancer- and/or tissue-specific mRNA using peripheral blood from patients would be a powerful and non-invasive diagnostic method. The reliability of this method is based on the balance of sensitivity in detection and specificity for cancer. Results for the detection of PSA and AFP in peripheral blood might be reliable if the sensitivity of detection is reduced. However, the possibility of false-positive results in the highly sensitive RT-PCR must be taken into consideration. The establishment of a molecular diagnostic system to detect circulating cancer cells using RT-PCR for more tumor- and tissue-specific mRNA would have an impact on clinical cancer research.

Acknowledgments

This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Education, Sciences, Sports and Culture and from the second term Comprehensive 10-year Strategy for Cancer Control and Cancer Research from the Ministry of Health and Welfare of Japan. We thank Drs T. Todoroki, E. Ueno and H. Hara for collecting samples and for helpful discussions and Ms A. Kikukawa for helping with DNA sequencing.

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Received July 13, 1998; accepted August 31, 1998
For reprints and all correspondence: Kazuhiko Uchida, Department of Biochemistry and Molecular Oncology, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan. E-mail: kzuchida{at}md.tsukuba.ac.jp
Abbreviations: AFP, [alpha]-fetoprotein; PSA, prostate-specific antigen; RT-PCR, reverse transcriptase polymerase chain reaction; HCC, hepatocellular carcinoma; AGPC, acid guanidinium thiocyanate-phenol-chloroform; MuLV, murine leukemia virus

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