Disturbed expression of the anti-apoptosis gene, Survivin, and EPR-1 in hematological malignancies

Leukemia Research 24 (2000) 965 – 970 www.elsevier.com/locate/leukres

Disturbed expression of the anti-apoptosis gene, Survivin, and EPR-1 in hematological malignancies Isao Shinozawa, Koiti Inokuchi *, Ichiji Wakabayashi, Kazuo Dan Di6ision of Hematology, Department of Internal Medicine, Nippon Medical School, 1 -1 -5 Sendagi, Bunkyo-ku, Tokyo 113 -8603, Japan Received 16 March 2000; accepted 11 April 2000

Abstract Survivin is a newly discovered inhibitor of the apoptosis protein, IAP, expressed during development and in human cancers. The effector cell protease receptor-1 (EPR-1) gene is oriented in the opposite direction on the same DNA double strand. Thus, the Survivin and EPR-1 (Survivin/EPR-1) genes exist in a head-to-head configuration. It is not clear whether mutual expression of the Survivin/EPR-1 genes occurs in both normal cells and cancer cells. Here, we investigated the mutual expression of the Survivin/EPR-1 genes in 12 normal peripheral blood (PB) specimens, seven normal bone marrow (BM) specimens, five lymph node (LN) specimens, and seven leukemic cell lines, and 27 patients with malignant lymphoma (ML), four with acute lymphocytic leukemia (ALL), three with acute myelocytic leukemia (AML), and four with chronic myelocytic leukemia in blastic crisis (CML-BC). Using Northern blot analysis, small amounts of EPR-1 mRNA were detected in normal PB, normal BM and LN specimens, but no Survivin mRNA was detected. However, Survivin mRNA was detected in two of the 12 normal PB, six of the seven normal BM and one of the five LN specimens using reverse transcription and polymerase chain reaction (RT-PCR). Expression of both the Survivin and EPR-1 genes was detected in six of the seven cell line samples by Northern blot, and in all of them by RT-PCR. Mutual expression of the Survivin and EPR-1 genes was detected in three of the four CML-BC samples, 15 of the 27 ML samples, two of the four ALL samples, and all three AML samples using the RT-PCR method. No EPR-1 expression with or without Survivin expression was clearly detected in eight of the nine diffuse large B-cell lymphoma (DLB) specimens, two of the six follicular center lymphoma specimens, one of the four specimens of nodular sclerosis of Hodgkin’s lymphoma, two of the four ALL specimens or one of the four CML-BC specimens. The data presented here show that disrupted expression of the Survivin/EPR-1 genes occurred in many kinds of hematologically malignant cells. This may be of biological importance. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: IAP gene; Survivin gene; EPR-1 (effector cell protease receptor-1); Hematological malignancies; Malignant lymphoma; Leukemia; RT-PCR (reverse transcription and polymerase chain reaction)

1. Introduction Regulated programmed cell death (apoptosis) preserves normal homeostasis and organ morphogenesis [1,2]. Aberrations of this process participate in various human diseases and may contribute to cancer by abnormally prolonging cell viability with accumulation of transforming mutations [3]. A novel member of the inhibitor of apoptosis protein, IAP, gene family, designated Survivin [4], was recently identified by hybridization screening of human genomic

* Corresponding author. Tel.: + 81-3-38222131; fax: +81-356851793.

libraries with the cDNA of a factor Xa receptor, effector cell protease receptor-1 (EPR-1) [5]. Unlike all other IAP [6,7], Survivin contained a single baculovirus IAP repeat and no RING finger [8–11]. Intriguingly, the Survivin non-coding strand was extensively complementary to the cording strand of EPR1. This suggests a potential functional interaction between these complementary transcripts [5]. The Survivin gene was selectively expressed during development but not in normal adult tissue in vivo [4]. However, Survivin becomes prominently expressed in transformed cell lines and in most cancers of the lung, colon, pancreas, prostate and breast [4]. The mutual relationship and expression of these reciprocal genes, the Survivin and EPR-1 (Survivin/EPR-1) genes, in a head-

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to-head configuration [12] remains unknown in both normal cells and hematological malignancies. Here, we analyzed mutual expression of the Survivin/ EPR-1 genes in order to elucidate the etiological significance.

2. Materials and methods

2.1. Cell samples Cells were obtained after informed consent from bone marrow (BM) aspirates, peripheral blood (PB) or lymph nodes (LN) of 38 patients with hematological malignancies at the time of diagnosis (Table 1). These included 22 non-Hodgkin’s lymphoma (NHL) (six follicular center lymphoma, nine diffuse large B-cell lymphoma (DLB), five peripheral T-cell lymphoma, one Burkitt’s lymphoma and one high-grade B-cell lymphoma), five Hodgkin’s disease (HD) (four nodular sclerosis and one lymphocytic predominance), four acute lymphocytic leukemia (ALL), three acute myelocytic leukemia (AML) and four chronic myelocytic leukemia in blastic crisis (CML-BC) [13]. A total of seven human cell lines, K562, M12, HL60, HEL, MY, OM9; 22 and TK from hematological malignancies were also examined [14,15]. Samples consisting of 12 normal PB-mononuclear cells (MNC) and seven normal BM were used as controls. Control LNs were obtained

from five patients with benign lymphadenopathy by possible viral infection.

2.2. Northern blot analysis The total RNA (tRNA) was extracted from cell samples with an RNA zol kit (Biotec Laboratories, Houston, USA). The integrity of the RNA samples was determined by agarose gel electrophoresis, and degraded RNA samples were rejected. tRNA was hybridized with a Survivin/EPR-1 HL probe or a control b-actin probe by our original methods described previously [16]. The Survivin/EPR-1 HL probe, which was generated from Survivin cDNA by the polymerase chain reaction (PCR) method, was subjected to Northern blot analysis.

2.3. Preparation of Sur6i6in/EPR-1 HL probe A probe, which was able to detect Survivin mRNA and EPR-1 mRNA simultaneously by Northern blot analysis, was made by primer A (5%-TGCCTGGCAGCCCTTTCTCAA-3%, corresponding to Survivin residues 2834–2854 and EPR-1 residues 1071–1091) and primer B (5%-TCAATCCATGGCAGCCAGCT-3%, corresponding to Survivin residues 12025–12041 and EPR-1 residues 689–706) (Fig. 1). Complementary DNA (cDNA) was prepared from 200 ng of total RNA using a random 9-mer oligonucleotide primer. PCR was per-

Table 1 Expression of the EPR-1 gene and Survivin genea Tested material (n)

Northern blot EPR-1+

Normal (24) Normal PB (12) Normal BM (7) LN (5)

RT-PCR Survivin+

EPR-1+

Survivin+

24 12 7 5

0 0 0 0

24 12 7 5

9 2 6 1

7

6

7

7

NHL (22) Follicular center lymphoma (6) Diffuse large B-cell lymphoma (9) Peripheral T-cell lymphoma (5) Burkitt’s lymphoma (1) High-grade B-cell lymphoma (1)

1 0 ND ND 1

1 0 ND ND 1

4 1 5 1 1

4 5 5 1 1

HD (5) Nodular sclerosis (4) Lymphocyte predominant (1)

ND ND

ND ND

4 1

4 1

2 3 2

4 3 2

2 3 3

4 3 4

Cell lines (7)

ALL (4) AML (3) CML-BC (4)

a Numbers are positive cases. Numbers in parentheses are analyzed cases. Lymphoma was classified using the ‘Revised European-American Lymphoma Classification’ [13]. ALL, acute lymphocytic leukemia; AML, acute myelocytic leukemia; BM, bone marrow; CML-BC, chronic myelocytic leukemia in blastic crisis; EPR-1+, EPR-1 positive; HD, Hodgkin’s disease; LN, lymph node; ND, not done; NHL, non-Hodgkin’s lymphoma; PB, peripheral blood; Survivin+, Survivin positive.

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using an ABI sequencer with dye terminators (Perkin Elmer, Warrington, UK). All sequences were confirmed in both orientations. The Survivin/EPR-1 HL probe was the collected RT-PCR insert digested by Apa I and Not I.

Fig. 1. Map of the Survivin gene and relationship with EPR-1/Survivin reading frames. Solid numbered boxes, exons; hatched box, CpG island; cross-hatched box, EPR-1 regulatory intron removed to generate the mature 1.3-kb EPR-1 transcript. Small bars indicate primers used for the Survivin and EPR-1 mRNAs.

Fig. 2. Sensitivity of Survivin or EPR-1 gene expression in RT-PCR assay. (A) Sensitivity of Survivin gene in RT-PCR assay and cycle numbers of PCR and PCR products (465 bp) of Survivin mRNA. Lane M: size marker fX 174 digested with Hae III and lDNA digested with Hind III; lane 1: 20 cycles; lane 2: 25 cycles; lane 3: 28 cycles; lane 4: 31 cycles; lane 5: 35 cycles. (B) Sensitivity of EPR-1 gene in RT-PCR assay and cycle numbers of PCR and PCR products (835 bp) of EPR-1 mRNA. Lane M: size marker; lane 1: 20 cycles; lane 2: 25 cycles; lane 3: 28 cycles; lane 4: 31 cycles; lane 5: 35 cycles.

formed using primers A and B, as described previously. After the RT-PCR products were separated on 2% agarose gel and stained with ethidium bromide, the amplified fragment was excised from the gel, electroeluted, purified with phenol, and precipitated with ethanol [17]. The fragments were subcloned into the EcoRV site of the pGEM-5Zf (+/ − ) cloning vector [18], which was then introduced to Escherichia coli HB101 cells. The transfected HB101 cells were plated on LuriaBeriani (LB) -ampicillin agar plates containing 5(X-Gal), bromo-4-chloro-3-indolyl-b-D-galactoside isotransferred to fresh LB-ampicillin agar plates containing X-Gal and isopropylthio-b-D-galactoside (IPTG), and then cultured overnight for secondary selection. White colonies were transferred into 150 ml of LB medium containing 50 mg/ml ampicillin and cultured at 37°C for 4 h. The cultured cells were sedimented by centrifugation, and the pellets were resuspended in 20 ml of water which was then heated at 98°C for 10 min. After centrifugation, the supernatants were amplified by PCR using the T7 or SP6 primer. The PCR products of three clones were sequenced

2.4. RT-PCR for detection of Sur6i6in and EPR-1 mRNAs RT-PCR for Survivin and EPR-1 gene expression was performed according to the previously described protocol [17] with a slight modification. cDNA synthesis was performed using a EPR-cDNA, 5%-AAGGCTGGGAGCCAGATGA-3% (EPR-1 residues 903–922) for EPR-1 cDNA preparation (Fig. 1). First, cDNA was synthesized with 500 ng of tRNA using each primer for 15 min at 42°C. The reaction solution was heated at 95°C for 15 s. The primer for Survivin cDNA was a Survivin-cDNA, 5%-ACGTGACAGATGTGTGAAGGTT-3% (Survivin residues 12958–12977). The RT-PCR reaction was performed with a slight modification of our original PCR procedure [18]. The PCR reaction for the Survivin and EPR-1 cDNAs was performed for 35 cycles for the Survivin and 31cycles for EPR-1 with 30 s at 94°C for denaturation, 30 s at 55°C for annealing, and 1 min at 72°C for extension using a DNA Thermal Cycler (Perkin-Elmer/Cetus, UK). The primers for PCR of Survivin cDNA were Survivin-5% and Survivin-3%, respectively, 5%-TTGGCAGGTGCCTGTTGAAT-3% (Survivin residues 12461–12480) and 5%-AGCCAGTCCCCCACAGCAT3% (Survivin residues 12907–12926). The primers for PCR of EPR cDNA were EPR-5% and EPR-3%, respectively, 5%-ATGACCTCCAGAGGTTTCCA-3%, (EPR-1 residues 37–56) and 5%-TCCGGTTGCGCTTTCCTTTC-3% (EPR-1 residues 853–872). Each amplified RT-PCR product was fractionated by electrophoresis on 2.0% agarose gel and visualized by staining with ethidium bromide (Fig. 2). Sequences of Survivin and EPR-1 RT-PCR products were confirmed after subcloning into the EcoRV site of pGEM-5Zf (+/−) cloning vector, as described above.

3. Results

3.1. Expression of the EPR-1 gene Total RNA from normal samples and cell lines were examined with Northern blot and RT-PCR analysis. Total RNA from hematological malignancies was examined mainly by RT-PCR analysis. Table 1 summarizes the results of the analysis. A small amount of EPR-1 mRNA was detected in all 24 normal PB, BM and LN specimens by Northern blot analysis, as shown in Fig. 3. RT-PCR analysis revealed that the EPR-1

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gene was clearly detected in all normal samples (24/24 samples) and all cell lines (7/7). However, decreased or absent expression of the EPR-1 gene was sometimes detected in hematological malignancies (Table 1, Fig. 4). In RT-PCR analysis, EPR-1 mRNA could not be

detected in eight of the nine DLB and two of the six follicular center lymphoma samples. EPR-1 mRNA was variably detected in HD, ALL, AML and CML-BC.

3.2. Expression of the Sur6i6in gene Table 1 summarizes the results of the analysis. In Northern blot analysis, the Survivin gene was not detected in all 24 normal samples. However, the Survivin gene was detected in many cell lines (6/7) and leukemias (9/9), as shown in Fig. 4. In RT-PCR analysis, detection of Survivin mRNA was improved. Survivin mRNA was detected in most of the hematological malignancies. However in normal PB and LN samples expression of the Survivin gene is reduced or absent.

3.3. Mutual expression analysis of the EPR-1 and Sur6i6in genes

Fig. 3. Expression of the Survivin and EPR-1 genes in Northern blot analysis. (A) Northern hybridization with a Survivin/EPR-1 HL probe, which was prepared by RT-PCR, identified two distinct and mutually expressed mRNAs of Survivin (1.9 kb) and EPR-1 (1.3 kb). (B) The lower panel shows the same ethidium bromide gel of Northern analysis. The upper band shows 28S ribosomal RNA and the lower band shows 18S ribosomal RNA. Lane 1: normal MNCs of PB; lane 2: MY cell; lane 3: normal MNCs of LN; lane 4: normal MNCs of BM; lane 5: K562 cell; lane 6: TK cell; lane 7: HEL cell; lane 8: HL60 cell; lane 9: OM 9; 22 cell.

Table 2 summarizes the results of the analysis. Mutual expression analysis of the EPR-1 and Survivin genes was mainly by the RT-PCR method. The mutual expression of the two genes was classified into four groups, Survivin-positive and EPR-1-positive (Survivin+ /EPR-1+), Survivin-negative and EPR-1-positive (Survivin − /EPR-1+), Survivin-positive and EPR-1-negative (Survivin+ /EPR-1− ) and Survivinnegative and EPR-1-negative (Survivin− /EPR-1−). In 12 normal PB, ten PB were Survivin− /EPR-1+ and two were Survivin+ /EPR-1+ . In seven normal BM, one BM was Survivin− /EPR-1+ and six were Survivin+ /EPR-1+. In five LN, four LN were Survivin− /EPR-1+ and one was Survivin+ /EPR-1+. Thus, normal PB and LN specimens tended to express the EPR-1 gene but not the Survivin gene. In contrast, normal BM tended to express both EPR-1 and Survivin gene. On the other hand, all seven cell lines and three AML patients were Survivin + /EPR-1+ . Many types of NHL were Survivin + /EPR-1+ . Although three of six follicular center lymphoma samples and eight of the nine DLB samples had other types of expression of Survivin/EPR-1 genes (Table 2), three of the four CML-BC patients were Survivin+ /EPR-1+.

4. Discussion

Fig. 4. RT-PCR analysis of Survivin and EPR-1 genes in the samples. Upper panel: Survivin products; middle panel: EPR-1 products; lower panel: b-actin as a control. A total of 25 cycles of PCR were performed for b-actin. Lane M: size marker fX 174 digested with Hae III; lane 1: HEL cell; lane 2: MY cell; lane 3: K562 cell; lane 4: AML; lane 5: CML-BC; lane 6: CML-BC; lane 7; ML (diffuse large B-cell); lane 8: ML Burkitt’s); lane 9: ML (diffuse large B-cell); lane 10: ML (diffuse large B-cell).

In the present study, we found EPR-1 expression with no Survivin expression (Survivin− /EPR-1+) in normal BM-MNCs, PB-MNCs and non-malignant LN cells when examined by Northern blot. However, RTPCR detected mutual expression of the Survivin/EPR-1 genes (Survivin+ /EPR-1+ ) in many of the BMMNCs. Survivin+ /EPR-1+ in BM-MNCs may reflect their immature characteristics. On the other hand, Sur-

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Table 2 Mutual expression analysis of EPR-1 and Survivin genes by RT-PCRa Tested material (n)

Survivin+/EPR-1+

Survivin−/EPR-1+

Survivin+/EPR-1−

Survivin−/EPR-1−

Normal (24) Normal PB (12) Normal BM (7) LN (5)

2 6 1

10 1 4

0 0 0

0 0 0

Cell lines (7)

7

0

0

0

NHL (22) Follicular center lymphoma (6) Diffuse large B-cell lymphoma (9) Peripheral T-cell lymphoma (5) Burkitt’s lymphoma (1) High-grade B-cell lymphoma (1)

3 1 5 1 1

1 0 0 0 0

1 4 0 0 0

1 4 0 0 0

HD (5) Nodular sclerosis (4) Lymphocyte predominance (1)

3 1

0 0

1 0

0 0

ALL (4) AML (3) CML-BC (4)

2 3 3

0 0 0

2 0 1

0 0 0

a Numbers in parenthesis are analyzed cases. Lymphoma was classified using the ‘Revised European-American Lymphoma Classification’ [13]. ALL, acute lymphocytic leukemia; AML, acute myelocytic leukemia; BM, bone marrow; CML-BC, chronic myelocytic leukemia in blastic crisis; HD, Hodgkin’s disease; LN, lymph node; NHL, non-Hodgkin’s lymphoma; PB, peripheral blood; Survivin+/EPR-1+, Survivin-positive and EPR-1-positive; Survivin−/EPR-1+, Survivin-negative and EPR-1-positive; Survivin+/EPR-1−, Survivin-positive and EPR-1-negative; Survivin−/EPR-1−, Survivin-negative and EPR-1-negative.

vivin +/EPR-1+ was common in all of the leukemia cell lines and some lymphomas. Thus, upregulated expression of the Survivin gene may play an important role in oncogenesis, as well as in development. Intriguingly, EPR-1 expression was not detected in eight of the nine DLB, two of the four ALL, two of the six follicular center lymphoma and one of the three CML-BC specimens. In lymphomas, upregulation of the Survivin gene was common, while downregulation of EPR-1 was specific to DLB and follicular center lymphoma. This might play a role in the oncogenesis of some lymphomas. In normal cells, EPR-1 was always expressed. Therefore, the absence of EPR-1 expression may be a marker of some hematological malignancies. Moreover, detection of Survivin expression by Northern blot analysis may be a marker of hematological malignancies, because none of normal PB, BM, or LN specimens revealed its expression. These findings on the expression of the EPR-1/Survivin genes suggest that an abnormal ratio of EPR-1 to Survivin protein may work toward the growth of malignant cells. Recent studies on IAP have attracted considerable attention because of their ability to suppress an evolutionarily conserved step in apoptosis [7], potentially involving direct caspase inhibition [19]. Dysregulation of the apoptosis pathway also participates in some human diseases, because inactivation by mutations of the neuronal apoptosis inhibitor protein (NAIP) of the IAP gene family contributed to spinal muscular atro-

phy [8], and NAIP was cytoprotective against cerebral ischemia in vivo [20]. More recently, this paradigm has been extended to cancer, with the identification of Survivin as a structurally unique IAP selectively expressed during development and in all the most common human cancers, but not in normal adult tissues in vivo [4]. Although the mutually exclusive distribution of EPR1 and Survivin in fetal tissues suggested a coordinated mechanism of gene regulation, it is not known whether an EPR-1 mRNA can down-regulate Survivin expression by acting as a natural antisense transcript [21–24]. The data of the present analysis suggest that coordination of the EPR-1 and Survivin gene expression may be independent. Inhibition of Survivin by forced expression of EPR-1 on HeLa cells reduced their viability [25]. However, there have been no reports that forced expression of Survivin induces cell proliferation and anti-apoptosis. This is the first report analysing mutual EPR-1 gene and Survivin gene expression in hematological malignancies . The expression patterns of the EPR-1/Survivin genes were disturbed in some hematological malignancies. The normal expression pattern, Survivin− and EPR-1+ , i.c., was scarcely detected in many types of hematological malignancies. Elucidation of the role of the Survivin and EPR-1 genes will facilitate knowledge of their participation in oncogenesis pathway in cancer and hematologic malignancies.

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