Stem Cells, Vol. 15, No. 3, 223-228,
May 1997
© 1997 AlphaMed Press
Decreased or Altered Expression of the FHIT Gene in Human Leukemias
Koichi Sugimoto,
Konagi Yamada,
Kiyoshi Miyagawa,
Hisamaru Hirai,
Kazuo Oshimi
Department of Hematology, Juntendo University School of Medicine and the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan
Key Words. Ap4A asymmetrical hydrolase • Exon skipping • FHIT gene • Inducible fragile site • Leukemia • RT-PCR • Tumor suppressor gene
Dr. Koichi Sugimoto, Department of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
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Abstract
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The FHIT (fragile histidine triad) gene on chromosome 3p14 is a candidate tumor suppressor gene, and its transcripts are shown to be abnormal in several human cancers. We examined 40 leukemia samples for the alterations of FHIT transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing. Intact FHIT mRNA was not detected in two patients with acute myeloid leukemia (AML) and in one patient with chronic lymphocytic leukemia (CLL). The three cases expressed only an aberrant FHIT mRNA lacking exons 3 to 6 (FHIT 
3-6 mRNA), which could encode a polypeptide of 13 amino acids. Southern blot analysis on two samples from these cases showed no rearrangements of the FHIT gene. Although intact FHIT mRNA was detected as the main band in the remaining 37 samples, 33 of them (14 of 14 AML, 11 of 13 chronic myeloid leukemia, five of five acute lymphocytic leukemia, and three of five CLL) expressed aberrant FHIT 3-6 mRNA. We barely detected the FHIT 3-6 mRNA in only one of 25 normal control samples. Our results suggest that loss of the normal FHIT function may be involved in the genesis of at least some human leukemias and that expression of aberrant FHIT transcripts is rather specific and frequent in leukemia samples.
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Introduction
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Recently, the human FHIT (fragile histidine triad) gene has been identified as a candidate tumor-suppressor gene at chromosome 3p14 because this gene spans the chromosomal region homozygously deleted in various epithelial cancer cell lines [1, 2]. Furthermore, the most common aphidicolin-inducible fragile site designated FRA3B, the breakpoint of the t(3;8) chromosomal translocation observed in a family with renal cell carcinomas, and an insertion site for the human papilloma virus type 16 were also mapped within the FHIT gene [1-4]. Expression of aberrant transcripts and loss of the normal transcript from the FHIT gene have been reported in esophagus, stomach, colon, lung and breast cancers [1, 4-6]. The FHIT gene belongs to the histidine triad gene family and encodes a protein with 69% similarity to Schizosaccharomyces pombe diadenosine 5',5'"-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase, which cleaves the Ap4A asymmetrically into ATP and AMP [1, 7]. Previous studies suggest that Ap4A may be implicated in various stress responses and regulation of DNA synthesis [8-10]. These observations indicate that loss or alterations of the FHIT gene function may play important roles in carcinogenesis.
Many lines of evidence support the view that malignant cell transformation is a multistep process that involves activation of proto-oncogenes and inactivation of tumor-suppressor genes [11]. Although considerable amounts of knowledge have been accumulated about the roles of tumor-suppressor genes in hematologic malignancies in the past several years, many things remain to be disclosed to understand the full picture of the genesis and progression of hematologic malignancies.
In the present report, we have examined 40 cases of myeloid and lymphoid leukemias for the alterations in structure and expression of the FHIT transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR) method and direct sequencing. Intact FHIT mRNA was not detected in three out of 40 leukemia patients. These cases were found to express only aberrant FHIT mRNA, which could not encode a functional protein.
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Materials and Methods
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Patients and Samples
Bone marrow samples from 40 patients, 16 with acute myeloid leukemia (AML), nine with chronic myeloid leukemia in chronic phase (CML-CP), four with CML in blast crisis (CML-BC), five with acute lymphocytic leukemia (ALL), and six with chronic lymphocytic leukemia (CLL), were collected after informed patient consent was obtained. The percentages of leukemic cells in the bone marrow samples were more than 60% in all cases (in cases of CML-CP, Ph1-positive cells were more than 60%). Bone marrow mononuclear cells were isolated with density sedimentation. As a negative control, peripheral blood mononuclear cells from 25 healthy volunteers were analyzed.
RT-PCR Method
The primers used in this study were shown according to the nucleotide numbers of the FHIT cDNA sequence published by Ohta et al. [1]. The sense primers were: HAP-OS, nucleotide (nt) -221 to -200; and HAP-IS, nt -192 to -173. The antisense primers were: HAP-OA, nt 594 to 573; and HAP-IA, nt 448 to 429. The RT-PCR was performed as follows: complementary DNA was synthesized from 1 µg of total cellular RNA from bone marrow mononuclear cells using 10 pmol of antisense primer HAP-OA and 200 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (GIBCO BRL; Gaithersburg, MD) in a 25 µl solution containing 200 µmol/l each of four dNTPs, 80 U of RNase inhibitor, 50 mmol/l Tris-HCl (pH 8.3), 75 mmol/l KCl, 10 mmol/l dithiothreitol (DTT), and 3 mmol/l MgCl2. The reaction was allowed to proceed for 60 min at 37°C and used as substrate for PCR. To the RT reaction solution, 25 µl of solution containing 250 µmol/l each of four dNTPs, 10 pmol of sense primer HAP-OS, 10 mmol/l of Tris-HCl (pH 8.3), 50 mmol/l KCl, and 5 U of recombinant Taq DNA polymerase (Takara; Kyoto, Japan) were added. First PCR was performed for 25 cycles of 94°C (1 min), 60°C (1 min) and 72°C (2 min). FHIT cDNA was generated using 1 µl (one-fifth) of RT-first PCR reaction solution by second PCR of 25 cycles (94°C 1 min, 60°C 1 min, and 72°C 2 min). The second PCR solution of 50 µl contained 10 pmol each of primers HAP-IS and HAP-IA, 125 µmol/l of four dNTPs, 10 mmol/l Tris-HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl2, and 2.5 U of Taq polymerase.
Sequencing
Normal-sized and aberrant DNA bands were excised from agarose gel and purified using Prep A Gene DNA Purification systems (Bio-Rad; Hercules, CA). Fifty ng of cDNA were directly sequenced using primers HAP-IS and HAP-IA by the dideoxy chain termination method on the 373A DNA sequencer (Applied Biosystems; Foster City, CA). Fifteen faint bands from normal volunteers were cloned using Original TA Cloning Kit (Invitrogen; San Diego, CA), and 0.5 µg of plasmid DNA was used for sequencing.
Southern Blot Analysis
DNAs extracted from bone marrow mononuclear cells of two patients without intact FHIT mRNA expression were digested with Eco RI and Bam HI, separated by electrophoresis on 0.7% agarose gel, and transferred to nylon filters. The hybridization probe was FHIT cDNA amplified by the second PCR.
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Results
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RT-PCR and Sequencing
Since previous studies detected aberrant FHIT transcripts which lack various regions between exons 4 and 9 [1, 4, 5], we amplified the FHIT cDNA spanning exons 2 to 9 by RT-PCR and examined them on agarose gel (Figs. 1 and 2). The amplified region covers 192 nucleotides of the 5'-untranslated region and the entire coding sequence. The expected-sized band of 640 bp corresponding to the intact FHIT mRNA was clearly amplified as the main band in all 25 normal control samples (Fig. 2, lanes N-N"). This normal-sized band was not detected in three of 40 leukemia samples (Fig. 2, lanes 1-3). The diagnoses of these three cases were two fresh AML (M3 and M4) and one advanced CLL (previously treated, Rai Stage IV). None of them had cytogenetic abnormalities of chromosome 3p. Only a much smaller band of 228 bp was amplified in all three samples. Direct sequencing revealed that the bands from the three samples have the same sequence and they correspond to an aberrant FHIT transcript which lacks exons 3 to 6 (FHIT 3-6 mRNA) (data not shown). Since the ATG codons of the FHIT open reading frame locate on exons 5 (original initiator codon), 6 and 9 (Fig. 1), FHIT 3-6 mRNA can encode only the FHIT C-terminal polypeptide of 13 amino acids. Therefore, functional FHIT transcripts were not detected in the three leukemia samples.
In all other 37 leukemia samples, normal-sized FHIT cDNA was amplified clearly as the main band (Fig. 2, lanes 4-9). Direct sequencing of these bands from 15 samples showed that they are intact FHIT cDNA (data, not shown). Only two sequence variants, a C to T transition at the third letter of codon 88 (alanine) and a T to C transition at the third letter of codon 94 (histidine), were detected. They seemed polymorphic because both of them were silent. Thirty-three of the 37 samples (14 of 14 AML, 7 of 9 CML-CP, 4 of 4 CML-BC, 5 of 5 ALL, and 3 of 5 CLL) had aberrant band(s) in addition to the intact FHIT cDNA (Table 1). Thirteen leukemia samples displayed both 228 bp and 281 bp additional bands (Fig. 2, lanes 4 and 5). Twenty leukemia samples had only the 228 bp additional band (Fig. 2, lanes 6 and 7). The remaining four samples (2 CML-CP and 2 CLL) did not have additional bands (Fig. 2, lanes 8 and 9). Direct sequencing of the 228 bp and 281 bp bands excised from agarose gel (eight sequencing analysis for each) revealed that they correspond to the FHIT 3-6 mRNA and an aberrant FHIT mRNA lacking exons 4 to 6 (FHIT 4-6 mRNA), respectively (Fig. 1). One CML-CP sample had another additional band of 356 bp together with the 228 bp band (data not shown). This band was shown to be an abnormal FHIT cDNA lacking the 3'-side 18-nucleotide sequence of exon 4, and exons 5 and 6. Agarose gel electrophoresis of a much larger amount of RT-PCR products from the 25 normal control samples (three times as much as usual, data not shown) revealed a very faint FHIT 3-6 cDNA band only in one sample. These results are summarized in Table 1.
A closer look at the pattern of RT-PCR products from 25 normal volunteers revealed that all of them had some additional faint band(s) of a little larger size together with the main band (Fig. 2, lanes N-N"). These faint bands were also detected in the 37 leukemia samples showing the normal-sized band. Fifteen of these bands from the normal volunteers were cut out from agarose gel, inserted into a plasmid and sequenced. They were alternatively spliced FHIT transcripts which contained an about 50- to 100-base insertion between exons 2 and 3, 3 and 4, or 4 and 5 (data not shown). For example, one of these transcripts had a 72-base insertion between exons 4 and 5 (data not shown). Another transcript had a 112-base insertion, a 5'-side 40-nucleotide sequence plus the 72-base insertion sequence, between exons 4 and 5. All of these insertions do not change the FHIT coding sequence.
Southern Blot Analysis
We examined the structure of the FHIT gene in two DNA samples available from the cases lacking the intact FHIT transcript (one AML (M3) and one CLL). Eco RI- and Bam HI-digested DNAs were hybridized with a probe spanning exons 2 to 9 of the FHIT cDNA (Fig. 3). Southern blot analysis showed no rearrangements of the FHIT gene in these two samples.
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Figure 3. Southern blot analysis of the FHIT gene on Eco RI-, and Bam HI-digested DNAs (A and B, respectively) from two leukemia samples lacking the normal FHIT mRNA (lane 1, AML (M3); lane 2, CLL). Lane N shows a normal control.
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Discussion
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The FHIT gene on chromosome 3p14 has emerged as a candidate tumor suppressor gene because it contains a target region of homozygous deletion in various cancer cell lines, and alterations of its transcripts are often observed in digestive tract, lung and breast cancers [1, 4-6]. In this study, bone marrow mononuclear cell samples from 40 patients with various leukemias were analyzed for the abnormalities of FHIT transcripts. Intact FHIT mRNA was undetectable in three patients. These three samples were shown to express only aberrant FHIT mRNA which could not encode the functional FHIT protein. The aberrant mRNA lacked exons 3 to 6, which may indicate internal alterations in the FHIT gene. This mode of inactivation of the FHIT gene contradicts the possibility that the gene might be simply located adjacent to the true target gene. Our observations agree with the hypothesis that loss of the normal FHIT function may be involved in the pathogenesis of at least some part of human leukemias.
Southern blot analysis showed normal configuration of the FHIT gene in two leukemia samples lacking intact FHIT mRNA expression. Negrini et al. reported that no rearrangements were found by Southern blot analysis in 41 breast cancer cell lines and primary tumors, although 12 of them exhibited lack of or alterations in FHIT transcripts [4]. KatoIII, a gastric cancer cell line, was shown to have deletions in the FHIT locus and expresses as only aberrant FHIT mRNA missing exons 4 to 7. However, all FHIT exons were intact in this cell line [1, 2]. Therefore, our observation might be explained as follows. Since the FHIT locus spans a huge chromosomal region of at least 500 kb, possible alteration(s) in the FHIT intron(s) to cause abnormal exon skipping could be too distant from the exons to be covered within the same restriction fragment in these two cases. Another possibility is that the resolution of the Southern blot analysis was not good enough to detect relatively small changes in the FHIT gene. Otherwise, abnormal exon skipping may be caused by really small genomic changes such as an intronic point mutation near the exon-intron boundaries in the FHIT gene. In fact, we found deletion of the 3'-side 18-nucleotide sequence of exon 4, and exons 5 and 6 in FHIT cDNA from one CML-CP sample, which seems to indicate a splice site mutation.
Of 37 leukemia samples having normal-sized FHIT cDNA, 33 samples were shown to express aberrant FHIT mRNA(s). The aberrant transcripts lack exons 5 and 6, which indicate that they can encode only the FHIT C-terminal polypeptide of 13 amino acids. Direct sequencing of 15 out of 33 normal-sized FHIT cDNA bands from these samples was shown to contain no mutations. Therefore, if the aberrant FHIT transcripts play direct roles in leukemogenesis in these cases, they should exert a dominant-negative effect on the normal FHIT mRNA. Abnormalities in the FHIT transcripts could be a result of genomic instability of leukemic cells, because one of the inducible fragile sites lies within the FHIT gene. In any way, 33 of 37 leukemia samples showing the normal-sized band expressed the FHIT 3-6 mRNA, although this transcript was barely detected only in one of 25 normal control samples. Expression of the FHIT 3-6 mRNA being rather specific and frequent in leukemia samples, it could be a useful marker for hematologic malignancies. In this regard, it seems very interesting to examine the FHIT transcripts in preleukemic states such as myelodysplastic syndrome.
Although we detected the FHIT 3-6 mRNA only in one of 25 peripheral blood mononuclear cell samples from normal volunteers, most of them were shown to express alternatively spliced FHIT transcripts which contain insertions of 50 to 100 nucleotides at various splice junctions of exons 2 to 5. These insertions do not change the coding sequence of FHIT mRNA. We found a 72-base insertion between exons 4 and 5 in a minor portion of FHIT transcripts from normal volunteers. In the previous reports, only the normal-sized RT-PCR product was detected in most samples from non-tumor colon or lung tissues, and the FHIT mRNA with a 72-base insertion between exons 4 and 5 was considered a tumor-specific transcript [1, 5]. The difference between the previous reports and our results may be due to a special feature of peripheral blood mononuclear cells, most of which are lymphocytes. Lymphocytes are the only cells in which somatic mutations occur and are supposed to be relatively prone to genetic changes. A minor population of normal lymphocytes might get some genetic changes at the fragile site within the FHIT gene and could express insertion-containing mRNAs. Recently published data reported the presence of various faint bands representing alternatively spliced FHIT transcripts in RT-PCR products from normal colon or brain tissues [6].
Normal FHIT mRNA was not detected in three leukemia samples. Two cases were fresh AML (M3 and M4) and one was advanced CLL (Rai Stage IV). Furthermore, 33 of 37 leukemia samples with normal-sized FHIT cDNA (14 of 14 AML, 7 of 9 CML-CP, 4 of 4 CML-BC, 5 of 5 ALL, and 3 of 5 CLL) expressed aberrant FHIT transcripts. These results suggest that abnormalities in the FHIT transcripts may occur in various types of leukemias and may have little subtype specificity. In this regard, the candidate tumor suppressor FHIT gene contrasts with p16INK4A and p15INK4B genes which are deleted specifically in lymphoid malignancies, especially of T-cell lineage [12, 13]. Involvement of p53 gene inactivation in the progression of various hematologic malignancies is well established [14]. We and others showed frequent inactivation of the p53 gene in various leukemia and lymphoma cell lines [15-17]. In the case of the FHIT gene, loss of the normal transcript was observed in only one of 15 leukemia cell lines (Sugimoto, unpublished result). The frequency of FHIT gene inactivation in leukemia samples is comparable with that in leukemia cell lines. Furthermore, two of the three cases lacking the normal FHIT transcript were non-treated leukemias. These observations indicate that inactivation of the FHIT gene may play a role at a relatively early stage of leukemogenesis in a small portion of various leukemias.
If both alleles of the FHIT gene are completely silent, RT-PCR may amplify the FHIT cDNA from the contaminating normal cells in the bone marrow samples. Therefore, some samples showing the normal-sized RT-PCR product could contain leukemic cells that entirely lack the FHIT gene expression. Inactivation of the FHIT gene might be more frequent than shown in this study.
The FHIT protein shows 69% similarity to S. pombe Ap4A asymmetrical hydrolase. Ap4A was proposed to mediate various stress responses [8, 9]. A drastic rise of intracellular Ap4A was also shown to correlate with the onset of DNA synthesis [10], and an Ap4A binding protein was reported to associate with DNA polymerase 
and stimulate it [18]. Thus, loss of the FHIT function could result in the constitutive accumulation of Ap4A, stimulation of DNA synthesis and uncontrolled cell proliferation. More extended study of the normal function of the FHIT protein and of its alterations in leukemic cells will give important insights into the mechanism of human leukemogenesis.
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Conclusion
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We examined alterations of the FHIT transcript in 40 leukemia samples by RT-PCR and direct sequencing. Intact FHIT mRNA was not detected in two AML patients and in one CLL patient. These three cases expressed only aberrant FHIT 3-6 mRNA, which encodes a polypeptide of only 13 amino acids. Southern blot analysis on two DNA samples available from these patients showed no gross rearrangements of the FHIT gene. Although intact FHIT mRNA was detected as the main band in the remaining 37 samples, 33 of them (14 of 14 AML, 11 of 13 CML, 5 of 5 ALL, and 3 of 5 CLL) expressed the aberrant FHIT 3-6 mRNA. We detected the FHIT 3-6 mRNA only in one of 25 normal control samples. Our results suggest that loss of the normal FHIT function may be involved in the genesis of at least some part of human leukemias and that expression of the aberrant FHIT transcripts is a rather specific and frequent event in leukemia cells.
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Acknowledgments
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We thank Drs. Seishi Ogawa, Naoto Hirano and Tsuyoshi Takahashi (the Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo) and Dr. Mikio Nishizawa (Department of Medical Chemistry, Kansai Medical University) for their technical advice. We also thank Dr. Akihiro Shimosaka (Pharmaceutical Division, Kirin Brewery Co., Ltd.) for his general support for this work.
Supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare and from the Ministry of Education, Science and Culture in Japan.
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References
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Ohta M, Inoue H, Cotticelli MG et al. The FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancers. Cell 1996;84:587-597.[Medline]
-
Kastury K, Baffa R, Druck T et al. Potential gastrointestinal tumor suppressor locus at 3p14.2 FRA3B site identified by homologous deletions in tumor cell lines. Cancer Res 1996;56:978-983.[Abstract/Free Full Text]
-
Wilke CM, Hall BK, Hoge A et al. FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites. Hum Mol Genet 1996;5:187-195.[Abstract/Free Full Text]
-
Negrini M, Monaco C, Vorechovsky I et al. The FHIT gene at 3p14.2 is abnormal in breast carcinomas. Cancer Res 1996;56:3173-3179.[Abstract/Free Full Text]
-
Sozzi G, Veronese ML, Negrini M et al. The FHIT gene at 3p14.2 is abnormal in lung cancer. Cell 1996;85:17-26.[Medline]
-
Thiagalingam S, Lisitsyn NA, Hamaguchi M et al. Evaluation of the FHIT gene in colorectal cancers. Cancer Res 1996;56:2936-2939.[Abstract/Free Full Text]
-
Huang Y, Garrison PN, Barnes LD. Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family. Biochem J 1995;312:925-932.
-
Baker JC, Jacobson MK. Alteration of adenyl dinucleotide metabolism by environmental stress. Proc Natl Acad Sci USA 1986;83:2350-2352.[Abstract/Free Full Text]
-
Baker JC, Ames BN. Alterations in levels of 5'-adenyl dinucleotides following DNA damage in normal human fibroblasts and fibroblasts derived from patients with xeroderma pigmentosum. Mutat Res 1988;208:87-93.[Medline]
-
Weinmann-Dorsch C, Hedl A, Grummt I et al. Drastic rise of intracellular adenosine(5')tetraphospho(5')adenosine correlates with onset of DNA synthesis in eukaryotic cells. Eur J Biochem 1984;138:179-185.[Medline]
-
Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet 1993;9:138-141.[Medline]
-
Hebert J, Cayuela M, Berkeley J et al. Candidate tumor-suppressor genes MTS1 (p16INK4A) and MTS2 (p15INK4B) display frequent homozygous deletions in primary cells from T- but not from B-cell lineage acute lymphoblastic leukemias. Blood 1994;84:4038-4044.[Abstract/Free Full Text]
-
Ogawa S, Hangaishi A, Miyawaki S et al. Loss of the cyclin-dependent kinase 4-inhibitor (p16; MTS1) gene is frequent in and highly specific to lymphoid tumors in primary human hematopoietic malignancies. Blood 1995;86:1548-1556.[Abstract/Free Full Text]
-
Imamura J, Miyoshi I, Koeffler HP. p53 in hematologic malignancies. Blood 1994;84:2412-2421.[Free Full Text]
-
Sugimoto K, Toyoshima H, Sakai R et al. Frequent mutations in the p53 gene in human myeloid leukemia cell lines. Blood 1992;79:2378-2383.[Abstract/Free Full Text]
-
Cheng J, Haas M. Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines. Mol Cell Biol 1990;10:5502-5509.[Abstract/Free Full Text]
-
Farrell PJ, Allan GJ, Shanahan F et al. p53 is frequently mutated in Burkitt's lymphoma cell lines. EMBO J 1991;10:2879-2887.[Medline]
-
Baxi MD, McLennan AG, Vishwanatha JK. Characterization of the HeLa cell DNA polymerase -associated Ap4A binding protein by photoaffinity labeling. Biochemistry 1994;33:14601-14607.[Medline]
accepted for publication February 20, 1997.
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Abstract
Introduction
