Dr Sheng-Fung Lin Division of Haematology-Oncology, Department of Internal Medicine, Kaohsiung Medical College Hospital, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan.
Recently, a tumour susceptibility gene, TSG101, has been identified at chromosome 11p15. A large intragenic deletion of this gene has been demonstrated in primary breast tumours. To evaluate the role of the TSG101 gene in leukaemia, bone marrow and/or peripheral blood from 68 acute myeloid leukaemia patients, five haemopoietic cell lines (HL60, U937, Raji, KG-1, K562) and 30 normal controls were analysed by reverse transcription of the TSG101 mRNA, followed by PCR amplification and sequencing of the products. The results showed aberrant TSG101 transcripts in 24/68 (35%) acute myeloid leukaemia (AML) patients, all of the cell lines (100%) and 3/30 (10%) normal controls. Our study indicated that the abnormal transcripts may have resulted from aberrant RNA splicing as evidenced by these aberrant transcripts. Also, normal full-length transcripts were present in all specimens examined. The aberrant transcript occurred more frequently in the AML and cell lines. However, because aberrant transcripts of TSG101 were also found in the normal controls, the role of TSG101 as a tumour suppressor gene should be evaluated carefully.
Increasing evidence has indicated that the tumourigenesis of malignancies is the result of a step-wise accumulation of mutations affecting both oncogenes and tumour suppressor genes ( Vogelstein et al, 1988 ; Knudson, 1989). In leukaemia, nonrandom karyotype changes have important clinical and diagnostic implications, and the common genetic abnormalities are chromosomal translocations juxtaposing genes to activate oncogenes or creating novel fusion genes with the production of chimaeric proteins ( Rabitts, 1994; Cline, 1994). However, some leukaemias seem to involve the mechanism of tumour suppressor genes although none of these genes have been mapped ( Knudson, 1993). Several reports have shown the involvement of chromosome 11p15 in acute myeloid leukaemia (AML) ( Mitelman, 1994; Nakamura et al, 1996 ; Huang et al, 1997 ), which has suggested that 11p15 might be affected in myeloid leukaemia.
The mouse tsg101 gene is a recently identified gene in which homozygous functional disruption induces cell transformation in mouse fibroblasts and forms metastatic tumours in nude mice ( Li & Cohen, 1996). The human homologue of the tsg101 gene, TSG101, was then isolated and mapped to 11p15.1-15.2, a region showing loss of heterozygosity in a variety of human malignancies and proposed to contain tumour suppressor gene(s) ( Li et al. 1997 ). The gene is predicted to encode a 380 amino acid protein which contains a proline-rich domain and DNA-binding motifs characteristic for a transcription factor and a coiled-coil domain shown to interact with stathmin ( Maucuer et al, 1995 ). Stathmin is a cytoplasmic phosphoprotein which is elevated in a variety of malignancies and proposed to coordinate and relay diverse signals regulating cell proliferation and differentiation ( Hanash et al, 1988 ; Sobel, 1991; Brattsand et al, 1993 ; Roos et al, 1993 ). Additionally, the N-terminal domain of TSG101 belongs to a group of apparently inactive homologues of ubiquitin-conjugating enzymes (E2), which may be dominant negative regulators of E2 activity in cell cycle control ( Koonin & Abagyan, 1997).
Recently, using a PCR-based strategy, large intragenic deletions of the TSG101 gene involving 50–95% of the genomic locus of the gene were identified in 7/15 primary human breast cancers ( Li et al, 1997 ). Because several reports showed the translocation of chromosome 11p15 in AML ( Roos et al, 1993 ; Mitelman, 1994) and an increase in the expression of stathmin ( Hanash et al, 1988 ), we utilized RT-PCR and direct sequencing to evaluate the role of the TSG 101 gene in AML.
MATERIALS AND METHODS
Patients and cell lines
Bone marrow and/or peripheral blood from 68 AML patients were collected at Kaohsiung Medical College Hospital from July 1992 to December 1996. Five haemopoietic cell lines (HL60, U937, Raji, KG-1, K562) and peripheral blood of 30 normal controls were also included in this study.
RNA extraction and reverse transcription
Total RNA was purified as described previously ( Lin et al, 1994 ) or using a commercial kit (TRIzoL® Reagent, Life TechnologiesTM, GIBCO BRL, N.Y., U.S.A.). The extracted RNA was treated with RNase-free DNase I (GenHunter Co., Brookline, Mass., U.S.A.). Reverse transcription was performed in a 20 μl final volume containing 1 μg RNA, 0.5 μg random primers (10 mers), 10 m M dithiothreitol (DTT), 0.5 m M dNTPs, 5 U RNasin (Promega, Madison, Wis., U.S.A.), 50 m M Tris-HCl (pH 8.3), 75 m M KCl, 3 m M MgCl2, and 200 U Moloney Murine Leukaemia Virus reverse transcriptase (Promega, Madison, Wis., U.S.A.). The reaction was first denatured for 5 min at 95°C and incubated at 37°C for 60 min. The reaction was then stopped by heat inactivation at 95°C for 5 min.
RT-PCR and cDNA sequencing
Nested PCR was carried out using primers, flanking the full coding sequence of the TSG101 cDNA, as described in Table I. 1 μl of cDNA was used for the first PCR amplification of 30 cycles, using primers P1u and P1d. The amplified products were diluted 20-fold, and 1 μl of the diluted product was used for a second round of PCR amplification for 35 cycles using two nested primers, P2u and P2d. PCR amplifications were carried out in a final volume of 25 μl containing 0.8 μM of primers, 50 μM of each dNTP, 1 × PCR buffer, and 1.25 U Taq DNA polymerase (Boehringer Mannheim GmbH, Mannheim, Germany). The PCR consisted of an initial denaturation at 95°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 2 min, using a PCR Thermocycler (Hybaid Ltd, U.K.). The nested PCR products were analysed by electrophoresis on 1.5% agarose gel, and the DNA fragments were detected using ethidium bromide staining. All RT-PCR products were then subjected to direct sequencing analysis. 5–50 ng of purified DNA were sequenced using an fmol DNA sequencing system kit® (Promega, Madison, Wis., U.S.A.). The primers, P2u, p2d and P3u, as described in 1 Table I, were used for sequencing analysis. All the reactions were repeated at least three times with controls.
Table 1. Table I. Oligonucleotide primers for the study of the TSG101 gene.
To assess the fidelity of the PCR and to exclude nonspecific PCR products, we refined the PCR procedure with AmpliTaq GoldTMDNA Polymerase (Perkin-Elmer Co., N.J., U.S.A.) which is activated after a high temperature incubation step (95°C for 12 min). The results were the same as when Taq polymerase was used. We also designed the P3u primer ( 1 Table I) located more 3′ as permitted to clarify the aberrant transcripts observed in our specimens and the results were consistent with the original results.
RT-PCR and cDNA sequencing analysis
In this study we examined the expression of the TSG101 gene in 68 AML specimens, five haemopoietic cell lines, and 30 normal controls. Using primers flanking the TSG101 coding region, a normal-sized RT-PCR product with a complete coding region of TSG101 was observed in all of the specimens examined. In addition to the normal-sized TSG 101 transcript, bands of smaller size were detected in 24/68 (35%) AML specimens (Fig 1A). In the specimens with aberrant transcripts (24/68), most displayed a normal- and an abnormal-sized product. Of those cases displaying aberrant transcripts, 15 cases had one abnormal transcript, seven cases had two aberrant transcripts, and two cases showed four aberrant transcripts. Sequence analysis of these smaller-sized products revealed aberrant TSG101 transcripts (Fig 2). 13 different aberrant transcripts were found in these aberrant RT-PCR products. The deletion of nucleotides (nt) 154–1054 was the most common one and was noted in 13/24 AML specimens with aberrant transcripts ( 2 Table II). In these cases, the coiled-coil domain, specified by nucleotides 810–1022 of the transcripts, was deleted. Therefore the protein may lose the function of interaction with stathmin. In addition, another five aberrant transcripts showed deletion of nucleotides 142–953, nt 284–1054, nt 154–1039, nt 279–1218 and nt 295–1258, also resulting in the loss of the coiled-coil domain. Other aberrant transcripts lacking nucleotides 133–447, nt 133–638, nt 133–730, nt 127–670, nt 183–798, nt 243–558 and nt 284–638 are predicted to remove nucleotides upstream from the coiled-coil domain and to generate a frameshift leading to premature termination of the TSG101 protein. The results in detail are summarized in Table II and Fig 3.
Table 2. Table II. Aberrant TSG101 transcripts observed in AML.
All of the haemopoietic cell lines examined showed both normal- and aberrant-sized RT-PCR products, and they all had more than one aberrant transcript. The aberrant transcript lacking nucleotides 154–1054 was present in all of the cell lines (Fig 1B). Three of 30 (10%) normal controls exhibited both normal and smaller fragments (Fig 1C). One control had a deletion of nt 154–1054 and one had deletion of nt 133–447; both were found in our AML patients. However, deletion of nt 144–667 was found in one normal control, which was not found in our leukaemic samples. The result is consistent with the study of Gayther et al (1997 ).
We also sequenced the full-length cDNA in all the transcripts. No point mutations were found.
Recently, the candidate tumour suppressor gene TSG101 was identified at chromosome 11p15.1-15.2. Mutation of this gene in breast cancer has been frequently identified ( Li & Cohen, 1996; Li et al, 1997 ). Abnormalities at 11p15 have been identified frequently in breast cancers, and also in a variety of other cancers, including myeloid leukaemia ( Mitelman, 1994; Nakamura et al, 1996 ; Huang et al, 1997 ). Therefore, in order to elucidate the role of TSG 101 in leukaemogenesis, 68 AML cases, five haemopoietic cell lines, and 30 normal controls were studied.
In the current study we found aberrant TSG101 RT-PCR products in 35% of AML patients, 100% of the haemopoietic cell lines, and 10% of normal controls. This result is consistent with the study of Li et al (1997 ) in which aberrant transcriptions with different intragenetic deletion of TSG101 were frequently identified. It is interesting that the aberrant transcripts of TSG101 were a common feature in the samples analysed. Gayther et al (1997 ) also found aberrant transcripts in normal controls, in contrast to Li et al (1997 ). Two of the aberrant transcripts in our normal controls had the same deletion locations as the AML patients. The aberrant transcripts occurred more frequently in the AML specimens and cell lines. This result is consistent with studies by Spruck et al (1994 ) and Gayther et al (1997 ). This indicates that the TSG101 gene may play a role in the progression of leukaemia. However, PCR amplification of aberrant transcripts caused by less stringent pre-mRNA splicing in cancer cells compared to normal tissues should be considered ( Haber & Harlow, 1997).
Although the in vivo function of the TSG101 protein is not clear, sequence analysis revealed that the TSG101 cDNA contains a proline-rich domain and DNA-binding motifs characteristic of transcription factors. In addition, the TSG101 protein encoded a coiled-coil domain that interacts with stathmin, a cytoplasmic phosphoprotein previously implicated in tumourigenesis ( Hanash et al, 1988 ; Sobel, 1991; Brattsand et al, 1993 ; Roos et al, 1993 ). In our study, two cases showed more than two aberrant transcripts, but in the study of Li et al (1997 ) all cases displayed only one or two aberrant TSG101 transcripts. There were more aberrant transcripts in the current study than those found in the breast cancer study ( Li et al, 1997 ), but the most common aberrant transcript lacking nucleotides 154–1054 was the same as observed in breast cancers. This deletion in the TSG101 gene directly eliminated all or part of the segment of the coiled-coil domain, which is specified by nucleotides 810–1022. Other aberrant transcripts also involved the deletion of the coiled-coil domain (type I) or removed nucleotides upstream from the coiled-coil domain (type II) and were predicted to generate a frameshift leading to premature termination of the TSG101 protein (Fig 3).
In comparing this study with our previous one of FHIT in AML ( Lin et al, 1997 ), both genes had a high frequency of aberrant transcripts in the specimens examined. The normal-sized transcripts were present in all the AML specimens and cell lines. However, only five cases of AML had both TSG101 and FHIT aberrant transcripts. These results indicate that there is no close relationship between TSG101 and FHIT in AML.
The abnormal transcripts may result from the aberrant RNA splicing as evidenced by the occurrence of aberrant transcripts in the AML specimens, cell lines and normal controls, the presence of more than two truncated transcripts in individual specimens, and the presence of the normal full-length transcripts in all tumour samples and cell lines examined. The presence of the normal-sized transcripts in all the cell lines is definitely different from the findings of Li et al (1997 ) and Sozzi et al (1996 ), who argued that these non-deleted normal-sized transcripts in tumourous tissue might be caused by nontumour cells. Our results of TSG101 expression in AML is compatible with previous studies of breast cancer ( Lee & Feinberg, 1997; Steiner et al, 1997 ). However, because aberrant transcripts of TSG101 were also frequently found in the normal controls, the role of TSG101 as a tumour suppressor gene should be evaluated carefully.