Loss of FHIT Expression in Acute Lymphoblastic Leukemia1

Vol. 5, 2409–2414, September 1999 Clinical Cancer Research 2409

Cora Hallas, Maher Albitar, Jean Letofsky, encodes a 147-amino acid protein with in vitro diadenosine 1 3 -Michael J. Keating, Kay Huebner, and 5Ј,5ٞ-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 ␮g of total RNA in a 20-␮l 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 ␮g 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.

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 ␤-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 (5Ј33Ј, Boulder, CO) as described by the manufacturer. Poly(A) mRNA (3 ␮g) 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 ϫ 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 ␣ 32 with [ - P]dCTP by random priming. Prehybridization and with goat serum, the slides were incubated with ␣-Fhit rabbit hybridization were carried out in 50% formamide, 5ϫ SSPE, serum (Refs. 9 and 19; diluted 1:4000 in PBS with 1% BSA) 10ϫ 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 ␣-rabbit immunoglobulin. The immunoglobulin was de- 2ϫ 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 ␮g) was digested with restric- RESULTS ␮ tion enzymes EcoRV or HindIII in a 40- l reaction mix con- Fhit Protein Expression. Uncultured leukemic cell sam- ϫ 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 5ϫ Denhardt’s solution, 5ϫ SSPE. 1% phoblastoid cell lines and 5 PBL samples from healthy volun- SDS, and 0.1 mg/ml single-stranded DNA. Hybridized mem- teers were also investigated. branes were washed in 2ϫ SSC, 0.1% SDS, and decreasing Surprisingly, none of the lymphocyte samples showed any concentrations of SSC (down to 0.1ϫ SSC) for 20 min each. staining by immunocytochemistry using anti-Fhit rabbit serum, Western Blot Analysis. Cells from cell lines and PBLs although Fhit expression was detected by Western blotting and were washed in PBS and incubated in lysis buffer [250 mM RT-PCR (see next paragraph). Other tissues used as controls NaCl, 50 mM Tris (pH 7.5), 1 mM EDTA, 1% Triton X-100, 1 (the kidney cell line 293 and normal breast and prostate tissue) mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 10 ␮g/ml displayed clear cytoplasmic staining in the epithelial cells. In- each of leupeptin and pepstatin] for 60–90 min on ice. Lysates flamed tonsil tissue containing nonmalignant lymphocytes also were centrifuged for 15 min at 10,000 ϫ g, and the supernatants showed no staining in the B- or T-cell compartments, although were used for further investigation. Protein concentration was a weak staining was difficult to judge due to the small amount measured in a BCA protein assay (Pierce, Rockford, IL). Protein of cytoplasm in lymphoblastoid cells. The tonsil epithelium, on (50–100 ␮g) was separated in 15% SDS-PAGE gels and blotted the other hand, showed clear cytoplasmic staining (data not onto nitrocellulose Hybond ECL (Amersham, Piscataway, NJ). shown). These results suggested that hematopoietic cells express After blocking in 5% nonfat milk, the membrane was incubated low levels of Fhit relative to epithelial cells. with anti-Fhit rabbit serum (Refs. 9 and 19; diluted 1:4,000 in Expression of Fhit protein was detected by Western blot in PBS-Tween) for 1–2 h at room temperature. The secondary all lymphoblastoid cell lines and normal PBL samples (Table 1), antibody was antirabbit immunoglobulin labeled with horserad- whereas it was found only in one of eight ALL cell lines and one ish peroxidase (Amersham). The signal was detected using the of seven other hematological cancer cell lines. In the Burkitt’s ECL system (Amersham) as described by the manufacturer. The lymphoma cell line Daudi and the Jurkat cell line (T-ALL), a quality of the protein was confirmed by incubating the mem- very faint Fhit band could be detected only after a very long

Table 2 Summary of FHIT RNA and protein expression Fhit proteina mRNAb Primary ALLs 1 Ϫ 2 ϩ 3 ϩ 4 Ϫ 5 Ϫ 6 Ϫ 7 Ϫ Fig. 1 Expression of Fhit protein in ALLs. Western blot analysis of 8 ϩ protein from two lymphoblastoid cell lines (Lanes 1 and 2), two ALL 9 ϩ cell lines (Lanes 3 and 4), and primary ALL cases 1, 5, 13, and 16 10 Ϫ (Lanes 5–8) with anti-Fhit antiserum (a; shown after long exposure) and 11 Ϫ with tubulin (b). ALL cases 1 and 5 (Lanes 5 and 6) show very weak 12 Ϫ bands of Fhit protein. The sizes of two marker bands and the size of the 13 ϩ tubulin protein (in kDa) are indicated to the right. 14 Ϫ 15 Ϫ 16 ϩ 17 Ϫ 18 Ϫ cell lines or PBL samples, thus confirming a low level of Fhit ALL-derived cell lines protein expression in lymphocytes. ϪϪ RS4;11 In summary, expression of Fhit protein could be detected in MV4;11 ϪϪ BI4;11 ϪϪ only 24% of the malignant samples (including primary ALL Jurkat (ϩ)c (ϩ)c cases and tumor cell lines) but was detected in 100% of normal Molt-3 ϪϪ samples (lymphoblastoid cell lines and PBL samples; see Table d ALL-1 ϩ ND 1). This difference was highly significant using the ␹2 test (P Ͻ ϪϪ CEM 0.001). 697 ϪϪ Other hematopoietic neoplasia-derived cell lines FHIT mRNA Expression. RNA samples from seven JM ϪϪ ALL-derived cell lines, six cell lines derived from other hema- Sup T11 ϪϪ tological malignancies, and seven normal lymphoblastoid cell ϪϪ K562 lines were examined for the presence of FHIT mRNA by North- Daudi (ϩ)c (ϩ)c TMP-1 ϪϪ ern blotting. FHIT mRNA was found in all normal lymphoblas- MonoMAC ϩ ND toid cell lines but in only two of the neoplastic cell lines GM1500 ϪϪ investigated (Table 2 and Fig. 2). After a long exposure of the a As detected by Western blotting. blot, the ALL cell line Jurkat and the Burkitt’s lymphoma cell b As detected by Northern blotting. line Daudi each showed a weak band of FHIT mRNA, thus c The bands were weak. d confirming the results obtained by Western blotting. ND, not done. RT-PCR Analysis of FHIT Transcript Expression. All samples were also investigated for FHIT mRNA expression by nested and seminested PCR amplification. Samples in which normal FHIT transcripts could not be detected by RT-PCR did exposure of the blot. Of the primary ALLs, only 6 of 18 cases not show any FHIT mRNA by Northern blotting or Fhit protein showed robust expression of Fhit protein that was similar in by Western blotting, but in some samples, normal-sized PCR signal strength to the lymphoblastoid cell lines or normal PBL products could be found despite the absence of Fhit protein or samples, with the same amount of protein being loaded in every FHIT mRNA as detected by Northern blotting. case (cases 2, 3, 8, 9, 13, and 16; see Table 2). After a very long Amplification of exons 3–10 by nested PCR in all lympho- exposure of the blots, using the same conditions as described for blastoid cell lines and normal PBL samples showed a FHIT the cell lines, faint bands could also be observed in the remain- transcript of wild-type length. However, three of eight ALL cell ing 12 primary ALL cases (Fig. 1). Because the leukemic cell lines and the immunoblastic lymphoma cell line JM displayed samples are contaminated with normal PBLs, a low level of Fhit no FHIT transcript at all, and in the CML cell line K562, only expression is probably due to the nonneoplastic cells in these a smear of numerous aberrant bands was found. The other cell cases. Interestingly, Fhit expression was found only in B-cell lines, including the lymphoblastoid cell lines and three of five leukemias and not in any of the six T-cell leukemias. The PBL samples, showed one to three faint aberrant bands in significance of this finding was confirmed using Fisher’s exact addition to the normal-sized band. In only one AML cell line test. Therefore, loss of Fhit expression seems to be more com- (TMP-1) with a t(9:11) chromosome translocation, the normal mon in T-cell leukemias than in B-cell leukemias. Fhit expres- band was the only FHIT RT-PCR product detected. Repeated sion in normal lymphocyte samples was lower than in the amplification of FHIT cDNA from the primary ALL cases did kidney cell line 293 used as a positive control, because 25 ␮gof not necessarily reveal the same products in each experiment. A protein from the 293 cell line gave a signal for Fhit protein total of 16 of 18 ALL cases displayed the normal-sized tran- similar to the signal from 50 ␮g of protein from lymphoblastoid script, but in 2 cases (cases 10 and 11), various attempts of FHIT

Fig. 2 a, expression of FHIT mRNA by Northern blotting. Poly(A) mRNA from lymphoblastoid and leukemia/lymphoma cell lines was hybridized with a FHIT cDNA probe spanning exons 2–9. Cell line names are shown at the top, and the size of FHIT mRNA is indicated to the right. FHIT bands can only be seen in the normal lymphoblastoid cell lines (indicated by lb) and are weak in Jurkat (ALL) and Daudi (Burkitt’s lymphoma) cells. b, ethidium bromide-stained gel photographed before the transfer of mRNA, illustrating quality and quantity of mRNA. The mRNAs shown in a were transferred from this gel.

amplification from two different cDNA preparations revealed the normal transcript in some of the reactions, but in others, no amplification product was found. ␤-Actin PCR of these samples revealed no sign of degradation or poor quality of the cDNA. This result can be explained by a very low amount of FHIT cDNA in the samples. Most of the ALL cases showed various faint, shorter, aberrant bands that were not reproducible with regard to occurrence and length in repeated amplifications. Sequencing of aberrant bands from cell lines or primary ALL Fig. 3 Expression of FHIT mRNA by seminested RT-PCR. FHIT samples revealed that in most cases, complete exons were miss- RT-PCR products in primary ALL cases 6, 14, 15, and 16 (Lanes 1–4), two PBL samples (Lanes 5 and 6), AML cell line TMP-1 (Lane 7), ALL ing, most often exons 4–7 or 4–8. cell line CEM (Lane 8), and two lymphoblastoid cell lines (Lanes 9 and Sugimoto et al. (22) reported aberrant transcripts most 10). The sizes of the marker bands (Lanes M; in bp) are indicated to the often lacking exons 3–6 in their leukemia cases. Because these right. aberrant transcripts would not be detected by our PCR with primers placed in exon 3, we developed a seminested PCR amplifying exons 2–10. The five cell lines displaying no normal-sized FHIT transcript in the nested PCR did not show it by lignant cell lines, and four primary ALL cases were in accord- this approach either. The primary ALL cases 10 and 11 again ance with the published cDNA sequences in each case. showed alternatively positive and negative results for normal- Polymorphic transcripts missing 11 bp at the beginning of exon sized FHIT cDNA. As in the nested approach, one to three 10 were detected in normal-sized cDNA as well as in aberrant additional aberrant bands were found in all cell lines except products; the missing 11 bp do not affect the open reading TMP-1 (shown in Fig. 3), which displayed only the normal- frame. No correlation was detected between the occurrence of sized band, and K562 ,which showed only a smear of aberrant aberrant transcripts and the lack of Fhit protein, unlike results in transcripts. By this approach, the aberrant bands were often as solid tumors, where a good correlation was observed between strong as the normal-sized bands, and sequencing revealed that the detection of aberrant FHIT RT-PCR products and the ab- the most prominent aberrant bands lacked exons 3–6 or 3–7, sence of the protein (9, 13). mostly accompanied by fainter bands lacking exons 4–7. Most The FHIT Locus. We did not detect genomic rearrange- of the primary ALL cases and three of five PBL samples showed ments in any of the primary cases or cell lines by Southern aberrant RT-PCR products. blotting using a FHIT cDNA probe encompassing exons 3–10. To summarize, the two different RT-PCR approaches revealed a lack of normal-sized FHIT transcripts in 5 of 15 (33%) DISCUSSION tumor cell lines but in none of the normal lymphoblastoid cell The FHIT gene at chromosome 3p14.2 is a tumor suppres- lines (Table 2), with the difference being significant (P Ͻ 0.05). sor gene that is deleted or inactivated in a large variety of Additionally, aberrant transcripts were found in all cell lines different human cancers. Deletion of both alleles is the most with the exception of one AML-derived cell line (TMP-1). frequently observed event, resulting in the loss of function of the The sequences of 10 randomly chosen normal-sized RT- gene (27). Introduction of FHIT sequences into tumor cell lines PCR products from three lymphoblastoid cell lines, three ma- suppressed their ability to form tumors in nude mice (19).

In this study, loss of Fhit protein expression was observed (26), although in our cases, these products were mostly nonre- very frequently in primary ALL cases and leukemia/lymphoma producible with regard to their occurrence and length. The exons cell lines. By Western blot analysis, only 24% of ALL cases or lacking in the most prominent aberrant bands usually included malignant cell lines expressed the Fhit protein, whereas all exon 3, confirming the results of Sugimoto et al. (22), who normal lymphoblastoid cell lines and PBL samples did. Loss of found RT-PCR products missing exons 3–6 to be the main Fhit expression was found in all T-cell leukemias investigated. aberrant transcripts in various leukemias. The low levels of Fhit protein detected in primary ALL cases Sugimoto et al. (22) did not report aberrant RT-PCR prod- after long exposure of the blots are probably derived from ucts in their normal PBL samples, but other studies reported contaminating normal lymphocytes in the samples. Northern aberrant products from lymphoblastoid cell lines (29), EBV- blotting of RNA from the cell lines confirmed the results ob- immortalized B-cells (30), and PBLs (24, 26, 30, 31), similar to tained by Western blotting. Normal FHIT mRNA was detected our results. Aberrant transcripts have also been reported in in all normal lymphoblastoid cell lines but in only 2 of the 13 various other normal tissues as well as in the tissue of the neoplastic cell lines investigated. These data strongly suggest corresponding tumor [liver (32), brain (33), cervix (34, 35), and that loss of Fhit expression occurs frequently in ALL samples, prostate (36)]. This aberrant mRNA may reflect reduced splic- especially those derived from T-cells. ing fidelity or may be due to rare rearrangement events occur- The results of two different RT-PCR approaches revealed a ring in the normal population similar to the t(14;18) and the lack of normal FHIT transcripts in 33% of the malignant cell t(9;22) chromosome translocations. These are observed specif- lines but in none of the normal lymphoblastoid cell lines and ically in follicular lymphoma and CML, respectively, but it is PBL samples. In the cell lines that did not show normal-sized also possible to detect these rearrangements by PCR in PBLs of cDNA by RT-PCR, Fhit protein or FHIT mRNA (by Northern healthy individuals (37, 38). blotting) was also not detectable. Genomic alterations that would explain the loss of FHIT Of the 18 primary ALL cases, 16 reproducibly showed nor- expression were not detected using a cDNA probe spanning exons mal-sized bands by RT-PCR. Because in 10 of these 16 cases only 3–10 in Southern hybridization experiments. This is in concordance traces of Fhit protein were found by Western blot, it seems likely with the results of Sugimoto et al. (22) and Peters et al. (26), who that the normal FHIT mRNA and protein may be derived from did not find genomic alterations in their cases either. Because the contaminating normal lymphocytes, detected more easily by the FHIT locus spans a chromosomal region of over 1 Mb, overlapping much more sensitive RT-PCR approach than by Western blot. deletions affecting exons of the gene may result in allelic alterations Accordingly, in six of the leukemia/lymphoma-derived cell lines, of FHIT without showing any genome rearrangement detectable by normal-sized FHIT RT-PCR products were found, but neither Fhit hybridization with cDNA (9). protein nor FHIT mRNA transcripts (by Northern blotting) were Taken together, the lack of Fhit protein and FHIT mRNA detected. Thus, detection of the full-length FHIT RT-PCR product in most of the ALL samples compared with the presence of the does not necessarily correlate with Fhit protein expression, because protein and mRNA in nearly all of the control samples indicates lack of normal transcripts in the main clone may be obscured by that the loss of Fhit expression may contribute to the pathogen- contaminating normal cells or by heterogeneity for the FHIT locus esis of a considerable fraction of ALL cases. Our data suggest within the tumor clone itself (6, 10). These results indicate that in that even when DNA or alterations of the RT-PCR product in hematopoietic malignancies, RT-PCR may be too sensitive to de- the protein coding region could not be observed, the Fhit protein tect loss of FHIT expression in the main clone, because even few and mRNA may be absent or dramatically reduced. interspersed normal cells will lead to a positive signal. Fhit protein expression is low in normal lymphoid cells ACKNOWLEDGMENTS relative to epithelial tissues and cells, which is consistent with We thank Rebecca Connor for excellent technical assistance and our failure to detect Fhit protein in lymphocytes using the Drs. Yuri Pekarsky and Raffaele Baffa for helpful discussions. immunocytochemistry approach. This result is also consistent with previous reports showing expression of Fhit in epithelial REFERENCES normal and tumor cells by immunohistochemistry, whereas in- 1. Ohta, M., Inoue, H., Cotticelli, M. G., Kastury, K., Baffa, R., filtrating B and T cells appear negative or nearly negative for Palazzo, J., Siprashvili, Z., Mori, M., McCue, P., Druck, T., Croce, FHIT (13, 28). Other investigators (22–26) reported few pri- C. M., and Huebner, K. The FHIT gene, spanning the chromosome mary leukemia cases lacking FHIT transcripts as detected by 3p14.2 fragile site and renal carcinoma-associated t(3:8) breakpoint, is RT-PCR. 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Cora Hallas, Maher Albitar, Jean Letofsky, et al.

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