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Loss of FHIT Expression in Acute Lymphoblastic Leukemia1

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Loss of FHIT Expression in Acute Lymphoblastic Leukemia1 Vol. 5, 2409–2414, September 1999 Clinical Cancer Research 2409 Loss of FHIT Expression in Acute Lymphoblastic Leukemia1 Cora Hallas, Maher Albitar, Jean Letofsky, encodes a 147-amino acid protein with in vitro diadenosine 1 3 Michael J. Keating, Kay Huebner, and 59,5--p ,p -triphosphate hydrolase activity (5). The physiolog- Carlo M. Croce2 ical function of Fhit is still unknown. Homozygous deletions, a hallmark of tumor suppressor Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, genes, have been observed in the FHIT gene in head and neck, Pennsylvania 19107 [C. H., J. L., K. H., C. M. C.], and The University of Texas M. D. Anderson Cancer Institute, Houston, Texas esophageal, gastric, colon, lung, and cervical cancers or cancer 77030 [M. A., M. J. K.] cell lines (1, 6–12), often including exon 5. Absence or alter- ation of FHIT transcription has been observed in head and neck, esophageal, gastric, pancreatic, lung, breast, kidney, and cervi- ABSTRACT cal carcinomas (4, 6, 7, 13–18) by RT3-PCR or Northern anal- Loss of expression of the FHIT tumor suppressor gene ysis. Siprashvili et al. (19) have shown that transfection of FHIT is common in epithelial malignancies such as lung, kidney, into tumorigenic cell lines suppresses tumorigenicity in nude esophageal, gastric, and cervical cancers. To assess the role mice, indicating that FHIT is a tumor suppresser gene. of FHIT in acute leukemias, we examined 18 primary acute Although chromosome 3 has not been reported to be a lymphoblastic leukemias (ALLs), 8 ALL-derived cell lines, 7 primary target of cytogenetic aberrations in leukemias, translo- cell lines from other hematological malignancies, 14 lympho- cations at 3p14 (20) and loss of heterozygosity at 3p (21) have blastoid cell lines, and 5 peripheral blood lymphocyte sam- been reported. Aberrant FHIT transcripts lacking various exons ples for expression of FHIT mRNA and protein by reverse between exons 3 and 9 have been found in acute and chronic transcription-PCR and Northern and Western blots. Fhit leukemias in addition to the wild-type transcript, but total loss of protein expression was detected in only 24% of primary FHIT mRNA expression has been detected infrequently (22– ALLs and leukemia/lymphoma cell lines, but it was detected 26). Nevertheless, absence of the Fhit protein has been detected in all lymphoblastoid cell lines and peripheral blood lym- in hematological malignancies (26). phocyte samples. Interestingly, Fhit protein expression was To assess the role of FHIT in ALL, we examined PBLs lost in all T-cell ALLs but was lost in only half of the B-cell from patients with ALL and ALL-derived cell lines and PBLs ALLs. Northern blotting of 7 normal lymphoblastoid cell from healthy volunteers and lymphoblastoid cell lines for DNA lines and 13 of the neoplastic cell lines confirmed the results integrity, transcription, and protein expression of the FHIT gene. obtained by Western blotting regarding FHIT expression. The high frequency of loss of Fhit expression in ALLs MATERIALS AND METHODS suggests that inactivating alterations at the FHIT locus con- Cells and Primary Leukemias. Cancer-derived cell tribute to development of the leukemias. lines were obtained from the American Type Culture Collection. All cell lines were maintained in RPMI 1640 containing 10% INTRODUCTION FCS and 0.1 mg/ml gentamicin. Uncultured leukemic cell sam- The FHIT gene at chromosome 3p14.2 spans over 1 Mb ples from 18 patients with ALL, including 6 T-cell and 12 B-cell and includes the FRA3B common fragile region, the t(3;8) leukemias, were obtained from The University of Texas M. D. (p14.2;q24) renal cell carcinoma-associated translocation, and a Anderson Cancer Center, and five samples of PBLs were ob- human papillomavirus integration site (1, 2). Distributed over tained from healthy volunteers. the FHIT genomic locus are 10 small exons encoding a 1.1-kb RNA Extraction, RT, and RT-PCR Amplification. mRNA. The first four exons are untranslated; exon 5 contains Total RNA was isolated from cell lines and PBLs using the the start codon, and exon 9 contains the stop codon. The FRA3B RNeasy Mini Kit (Qiagen, Valencia, CA) according to the fragile region encompasses over 500 kb surrounding exon 5, the manufacturer’s instructions, and RT was performed from 2–3 t(3;8) break lies in intron 3, and the human papillomavirus mg of total RNA in a 20-ml final volume of 20 mM Tris-HCl (pH integration site involves intron 4 (1, 3). The FHIT gene is 8.4), 50 mM KCl, 2.5 mM MgCl2,10mM DTT, 0.5 mM dNTPs, expressed at varying levels in most adult tissues (1, 4) and 0.5 mg of oligo(dT) primers, and 200 units of reverse tran- scriptase at 42°C for 60 min. The reaction was terminated at 70°C for 10 min. Nested RT-PCR amplifications were carried out as de- Received 4/22/99; revised 5/10/99; accepted 5/18/99. scribed previously (1). Additionally, seminested RT-PCRs were The costs of publication of this article were defrayed in part by the performed under the same conditions using primers 5D2 (exon payment of page charges. This article must therefore be hereby marked 2) and 3U2 (exon 10) in the first round and primers 5D2 and advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Core Grant CA 56036 and Grants CA 80677 and CA 39860 (to C. M. C.). 2 To whom requests for reprints should be addressed, at Kimmel Cancer 3 The abbreviations used are: RT, reverse transcription; ALL, acute Institute, BLSB, Room 1050, 233 South 10th Street, Philadelphia, PA lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic 19107. Phone: (215) 503-4645; Fax: (215) 923-3528. myeloid leukemia; PBL, peripheral blood lymphocyte. Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 1999 American Association for Cancer Research. 2410 FHIT Expression in Acute Lymphoblastic Leukemia 3U1 (exon 10) (1) in the second round of amplification. PCR Table 1 Expression of Fhit by Western blotting products were analyzed in a 1.5% agarose gel. Amplification No. of cases with b-actin primers was performed from every sample to confirm the quality of the cDNA. Positive Negative Sequence Analysis. PCR-amplified bands were excised Primary B-cell ALLs 6 6 from the gels, and DNA was extracted using Qiaquick Gel Primary T-cell ALLs 0 6 ALL cell lines 1 7 Extraction Kit (Qiagen) according to the manufacturer’s instruc- Hematol. neoplasia-derived cell lines 1 6 tions. DNA (25 ng) was directly sequenced using one of the PCR primers for cycle sequencing and analyzed in ABI 377 Total, neoplastic samples 8 (24%) 25 (76%) automated sequencers. Lymphoblastoid cell lines 14 0 Northern Blot Analysis. Total RNA from the cell lines Normal PBL samples 5 0 was obtained by extraction with Trizol reagent (Life Technol- ogies, Inc., Grand Island, NY) according to the manufacturer’s Total, nonneoplastic samples 19 (100%) 0 (0%) instructions. Poly(A) mRNA was isolated from 1 mg of total RNA using oligo(dT) cellulose spin columns (59339, Boulder, CO) as described by the manufacturer. Poly(A) mRNA (3 mg) was separated by electrophoresis in 0.8% denaturing agarose branes with anti-tubulin immunoglobulin (Neomarkers, Fre- gels, and the quality of the mRNA was checked visually under mont, CA) as primary antibodies instead of anti-Fhit serum. UV illumination. The mRNA was then transferred to nylon Immunocytochemistry. Cells from cell lines and PBL 3 membranes in 20 SSC. The membranes were hybridized with samples were washed in PBS and fixed in 4% PBS-buffered a cDNA probe consisting of exons 2–9 of the FHIT gene labeled formalin for 10 min before drying on glass slides. After blocking a 32 with [ - P]dCTP by random priming. Prehybridization and with goat serum, the slides were incubated with a-Fhit rabbit hybridization were carried out in 50% formamide, 53 SSPE, serum (Refs. 9 and 19; diluted 1:4000 in PBS with 1% BSA) 103 Denhardt’s solution, 2% SDS, and 0.1 mg/ml single- overnight at 37°C. They were then incubated with biotinylated stranded DNA at 42°C. Hybridized membranes were washed in goat a-rabbit immunoglobulin. The immunoglobulin was de- 23 SSC, 0.1% SDS, and in decreasing concentrations of SSC tected using the Vectastain ABC Reagent (Vector Laboratories, for 20 min each at 60°C. Burlingame, CA) according to the manufacturer’s instructions; Southern Blot Analysis. High molecular weight DNA the cells were counterstained with Harris’ Hematoxylin. from cell lines and PBLs was obtained using standard phenol- chloroform extraction. DNA (6 mg) was digested with restric- RESULTS m tion enzymes EcoRV or HindIII in a 40- l reaction mix con- Fhit Protein Expression. Uncultured leukemic cell sam- 3 taining 1 buffer supplied by the manufacturer and 20 units of ples from 18 ALL patients, 8 ALL cell lines, and 7 cell lines enzyme. The DNA was separated by electrophoresis in 0.7% derived from other hematological malignancies [2 AMLs, 1 agarose gels and blotted to nylon membranes using the Probe CML, 1 immunoblastic B-cell lymphoma, 1 T-cell lymphoma, 1 Tech 2 Oncor machine according to the manufacturer’s instruc- Burkitt’s lymphoma, and 1 tumorigenic lymphoblastoid cell line tions. The membranes were hybridized with the same probe as (GM1500–6TG-Oub)] were analyzed by immunocytochemistry the Northern blots. Prehybridization and hybridization were and Western blotting. As controls, 14 EBV-transformed lym- carried out at 65°C in 53 Denhardt’s solution, 53 SSPE. 1% phoblastoid cell lines and 5 PBL samples from healthy volun- SDS, and 0.1 mg/ml single-stranded DNA.
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