| | Microsatellite mutations of transforming growth factor-β receptor type II and caspase-5 occur in human precursor T-cell lymphoblastic lymphomas/leukemias in vivo but are not associated with hMSH2 or hMLH1 promoter methylationReceived 19 December 2001; accepted 22 April 2002. Abstract In solid cancers, defective DNA mismatch repair (MMR) is most commonly caused by hMSH2 or hMLH1 mutations, or epigenetic silencing of hMLH1 by promoter hypermethylation, and results in the acquisition of characteristic frameshift microsatellite mutations of mononucleotide repeats located within the coding regions of defined target genes. We previously identified hMSH2 mutations in T-cell lymphoblastic lymphoma (T-LBL) patient tumor samples and others have reported coding region microsatellite mutations in T-cell acute lymphoblastic leukemia (T-ALL) cell lines. Thus, while MMR gene mutations are known to occur in some human T-lymphoblastic tumors in vivo, it is still unknown if the coding region microsatellite mutations detected in human cell lines also occur in vivo or if hMLH1 or hMSH2 promoter hypermethylation contributes to defective MMR in these tumors. We analyzed the TGFβRII (A)10 and caspase-5 (A)10 coding region repeats in 16 human T-LBL/ALL patient tumor samples and identified six with microsatellite mutations in one or both repeats. There was no evidence of hMSH2 or hMLH1 promoter methylation as assessed by standard methylation specific PCR or by a novel temporal temperature gradient electrophoresis (TTGE) method that analyzed 25 and 30 CpG sites in the hMLH1 and hMSH2 promoters, respectively. Our results indicate that coding region microsatellite mutations characteristic of defective MMR occur in some human T-LBL/ALL in vivo but not as a consequence of hMLH1 or hMSH2 promoter hypermethylation. Furthermore, the identification of TGFβRII and caspase-5 coding region mutations in vivo implicates these genes in the pathogenesis of human T-LBL/ALL.
1. Introduction  Defective DNA mismatch repair (MMR) occurs in up to 20% of sporadic colorectal, gastric, pancreatic and endometrial cancers and most often results from hMSH2 or hMLH1 inactivating mutations or hMLH1 promoter methylation [1], [2], [3], [4], [5], [6], [7]. Such MMR defective solid cancers harbor frameshift mutations within mononucleotide microsatellites present in the coding regions of transforming growth factor-β receptor type II (TGFβRII) and other important target genes [8], [9], [10], [11]. Evidence is accumulating implicating defective MMR in the pathogenesis of T-cell lymphoblastic malignancies. Mice deficient in MSH2 develop T-cell lymphoblastic lymphomas (T-LBLs) associated with inactivating frameshift mutations within short mononucleotide runs present in the murine TGFβRII coding region [12], [13]. In humans, hMSH2 mutations have been identified in tumor samples from patients with T-LBL and both hMSH2 and hMLH1 mutations have been identified in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines [12], [14], [15]. Various studies of human cell lines have identified TGFβRII (A)10 and/or BAX (G)8 microsatellite mutations in B-ALL and T-ALL and a recent large survey of 91 human B-lymphoid, T-lymphoid and myeloid leukemia cell lines found the frequency of coding region microsatellite mutations within various target genes, including TGFβRII and BAX, to be highest in T-ALL [16], [17]. Presently, however, the evidence linking defective MMR with human T-cell lymphoblastic tumor pathogenesis in vivo is limited to the identification of hMSH2 mutations in some patient biopsy specimens. It is still not known if coding region microsatellite mutations occur in T-LBL/ALL in vivo or if MMR gene promoter hypermethylation contributes to defective MMR in these tumors. To investigate this further, we analyzed T-LBL and -ALL patient biopsy samples for coding region microsatellite mutations in TGFβRII and caspase-5, two defined gene targets of defective MMR in solid cancers in vivo, and for methylation of the hMLH1 and hMSH2 promoters. Our results indicate that TGFβRII and caspase-5 microsatellite mutations, that are characteristic of defective MMR, occur in some human T-cell lymphoblastic tumors in vivo but not as a consequence of hMLH1 or hMSH2 promoter hypermethylation. The identification of TGFβRII and caspase-5 coding region mutations in vivo may also indicate an important tumor suppressor function for these genes in some human T-LBL/ALL. 2. Materials and methods 2.1. Specimens Archival DNA from 10 cases of human T-LBL and six cases of human T-ALL was obtained from a bank of well characterized samples obtained from previously untreated patients at diagnosis and maintained at Tohoku University, Sendai, Japan (Table 1). All but two cases (T4 and T6) were from adult patients defined as ages 16 and older [18]. All cases had lymphoblastic morphology, expressed terminal deoxynucleotidyl transferase (TdT), expressed cytoplasmic and/or surface CD3 and clonal T-cell receptor gene rearrangements as assessed by Southern analysis. All T-LBL biopsy samples comprised sheets of malignant lymphoblasts with minimal intermingling normal cells and in all cases were obtained from patients found to be negative for bone-marrow infiltration by light microscopy. All T-ALL samples were obtained from unsorted bone-marrow aspirates containing greater than 90% lymphoblasts. Control DNA samples were randomly selected from a bank maintained at the University of Saskatchewan. The DNA source for these samples was peripheral blood mononuclear cells isolated by density centrifugation on Ficoll-Paque PLUS (Amersham Pharmacia Biotech Inc., Baie d’Urfe, PQ) from adult subjects who had been investigated for Factor V Leiden and found to be negative.  | Case | Disease | Age | Sex | White blood cella | Blast cells (%)b | Sitec | Immunophenotyped | |
| T1 | T-LBL | 44 | Male | 8.0 | NA | Mediastinal mass | CD1a−, CD4−, CD8−, CD7+, CD2+ CD5−, sCD3−, cCD3+, CD10−, TdT+ | |
| T2 | T-LBL | 23 | Male | 4.8 | 0 | Mediastinal mass | CD1a+, CD4−, CD8+, CD7+, CD2−, CD5+, sCD3+, CD10−, TdT+ | |
| T3 | T-LBL | 25 | Femae | 3.4 | 0 | Mediastinal mass | CD1a+, CD4+, CD8+, CD7+, CD2+, CD5+, sCD3+, CD10−, TdT+ | |
| T4 | T-LBL | 13 | Male | 6.2 | 0 | Mediastinal mass | CD1a−, CD4−, CD8−, CD7+, CD2−, CD5−, sCD3−, cCD3+, CD10−, TdT+ | |
| T5 | T-LBL | 28 | Male | NAe | NA | Mediastinal mass | CD1a−, CD4+, CD8−, CD7+, CD2+, CD5+, sCD3+, CD10+, TdT+ | |
| T6 | T-LBL | 14 | Female | 7.6 | 0 | Cervical lymph node | CD1a+, CD4+, CD8+, CD7+, CD2+, CD5+, sCD3+, CD10−, TdT+ | |
| T7 | T-LBL | 62 | Male | 6.8 | 0 | Pleural effusion | CD1a−, CD4−, CD8−, CD7+, CD2− CD5+, sCD3−, cCD3+, CD10−, TdT+ | |
| T8 | T-LBL | 26 | Male | 8.9 | 0 | Mediastinal mass | CD1a−, CD4−, CD8−, CD7+, CD2− CD5+, sCD3+ CD10+, TdT+ | |
| T9 | T-LBL | 16 | Male | 10.3 | <5 | Cervical lymph node | CD1a+, CD4+, CD8+, CD7+, CD2+, CD5+, sCD3+, CD10+, TdT+ | |
| T10 | T-LBL | 21 | Male | 6.2 | 0 | Pleural effusion | CD1a+, CD4+, CD8+, CD7+, CD2+ CD5+, sCD3−, cCD3+, CD10+, TdT+ | |
| T11 | T-ALL | 35 | Male | 12.3 | 78 | Bone-marrow | CD1a−, CD4−, CD8−, CD7+, CD2+, CD5+, sCD3−, cCD3+, TdT+, CD33+ | |
| T12 | T-ALL | 19 | Female | 26.9 | 90 | Bone-marrow | CD1a−, CD4−, CD8−, CD7+, CD2− CD5+, sCD3−, cCD3+, TdT+, CD33− | |
| T13 | T-ALL | 22 | Male | 1.7 | 48 | Bone-marrow | CD1a−, CD4−, CD8−, CD7+, CD2+ CD5−, sCD3−, cCD3+, TdT+, CD33+ | |
| T14 | T-ALL | 41 | Male | 30.5 | 81 | Bone-marrow | CD1a−, CD4+, CD8+, CD7+, CD2+ CD5+, sCD3+, TdT+, CD33− | |
| T15 | T-ALL | 24 | Female | 24.3 | 97 | Bone-marrow | CD1a−, CD4−, CD8−, CD7−, CD2+ CD5−, sCD3−, cCD3+, TdT+, CD33+ | |
| T16 | T-ALL | 30 | Male | 53.9 | 85 | Bone-marrow | CD1a−, CD4−, CD8−, CD7+, CD2+ CD5+, sCD3−, cCD3+, TdT+, CD33+ | | | | |
|
a
Peripheral blood white blood cell count (× 109/l).
b
Percentage of peripheral blood white blood cells that are blast cells.
c
Biopsy site from which T-LBL/ALL patient DNA was obtained.
d
All immunophenotypic markers with the exception of cytoplasmic CD3 (cCD3) represent surface antigen expression as detected by flow cytometric analysis of cell suspensions prepared from portions of tissue biopsies or bone-marrow aspirates. The expression of cCD3 was determined by immunohistochemical analysis of formalin fixed tissue.
e
NA, information not available. |
2.2. Analysis of the TGFβRII (A)10 and caspase-5 (A)10 repeats for microsatellite mutations Microsatellite mutations within the TGFβRII (A)10 repeat were identified by temporal temperature gradient electrophoresis (TTGE) and confirmed by sequencing. WinMelt software (Bio-Rad Laboratories Ltd., Mississauga, Ont.) was used to optimize the melting profiles of the wild-type TGFβRII (A)10 repeat (GenBank Primary Accession no. U52240) and mutant (A)9 and (A)11 repeats by the addition of GC clamps to the amplified PCR products. Vector NTI software (InforMax Inc., North Bethesda, MD) was used to design GC-clamp PCR primers flanking the repeat area and to select an internal hybridization probe (Table 2). Control plasmids containing TGFβRII (A)10, (A)9 or (A)11 repeats were constructed by cloning PCR fragments of genomic DNA from previously characterized human hematological cancer specimens using the Zero-Blunt cloning system (Invitrogen, San Diego, CA). High fidelity PCR amplification of DNA obtained from human T-LBL/ALL cases, control peripheral blood mononuclear cells and control plasmids was performed for 30 cycles at 95 °C for 1min, 49°C for 1min and 72°C for 2min using Pfu polymerase (Stratagene, La Jolla, CA). PCR products were electrophoresed on 6% polyacrylamide gels containing 7M urea in a DCode apparatus (Bio-Rad Laboratories Ltd., Mississauga, Ont.) at 130V for 8.5h in 1× Tris–acetate/EDTA buffer over a temperature range of 56–59°C at an average ramp rate of 0.35°C/h. Following electrophoresis, gels were transferred and fixed to nylon membranes, pre-hybridized in oligonucleotide hybridization buffer (10% PEG 8000, 1% SDS, 5× SSC) for 1h, hybridized to the internal probe labeled with  -dCTP in the same buffer overnight at 46°C then washed and exposed to a phosphorimaging screen. Differentially migrating PCR products were visualized and quantified using a Molecular Imager FX instrument equipped with Quantity One imaging software (Bio-Rad Laboratories Ltd., Mississauga, Ont.). TTGE screening results were confirmed by repeating the PCR and TTGE analysis, staining the gel with ethidium bromide and excising PCR product bands migrating in the wild-type (A)10 position and in mutant positions from positive patient samples. Excised bands were then purified using the Qiaquick PCR Purification System (Qiagen Inc., Mississauga, Ont.) and sequenced on an ABI Prism 310 Genetic Analyzer using dRhodamine Terminator Cycle Sequencing Ready Reaction kits with AmpliTaq DNA polymerase, FS (Applied Biosystems, Foster City, CA). |
a
Fw: forward (sense) primers; Rv: reverse (antisense) primers; M: primers specific for bisulfite modified, methylated DNA; U: primers specific for bisulfite modified, unmethylated DNA. For primer sets designed to distinguish methylated from unmethylated DNA following bisulfite modification, sequence differences between primers and unmodified DNA are in boldface type and potential sites of cytosine methylation within CpG dinucleotides are underlined.
b
nt: nucleotide position according to the GenBank accession numbers listed in Section 2.
c
Refers to the temperatures for optimal annealing of primer pairs and hybridization of oligonucleotide probes. |
The caspase-5 (A)10 repeat (GenBank Primary Accession no. AP001153) was screened for microsatellite mutations by direct sequencing. In this approach, PCR products showing sequence overlap at the distal end of the (A)10 repeat were deemed screen positive and were confirmed by cloning and sequencing. Vector NTI software (InforMax Inc., North Bethesda, MD) was used to select primers flanking the (A)10 repeat area (Table 2). High fidelity PCR amplification of DNA from human T-LBL/ALL cases and control peripheral blood mononuclear cells was performed for 30 cycles at 95, 49 and 72°C for 1min using Pfu polymerase (Stratagene, La Jolla, CA). PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen Inc., Mississauga, Ont.) and directly sequenced on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Products screening positive for caspase-5 (A)10 microsatellite mutations were confirmed by subcloning in the pCR-Blunt vector using the Zero-Blunt PCR Cloning Kit (Invitrogen, San Diego, CA). Ten clones were picked at random with at least two separate clones containing mutations within the (A)10 repeat required for confirmation. 2.3. Analysis of TGFβRII, hMSH2 and hMLH1 promoter regions for CpG methylation by methylation-specific PCR (MSP) DNA methylation within the CpG islands of the TGFβRII (GenBank Primary Accession no. U52240), hMSH2 and hMLH1 promoter regions was determined by MSP following sodium bisulfite treatment of genomic DNA [19]. Briefly, 10μg of genomic DNA was denatured in 0.3N NaOH at 37°C for 15min and incubated with 3M sodium bisulfite and 20mM hydroquinol at 55°C for 6h. Following this, DNA was purified using Wizard DNA Clean-Up Columns (Promega, Madison, WI, USA), incubated in 0.3N NaOH at 37°C for 15min, precipitated in ammonium acetate and ethanol, washed in 70% ethanol and re-suspended in water. The 5′-CpG island of TGFβRII was identified using the CpG Plot/CpG report utilities available from the European Molecular Biology Open Software Suite (EMBOSS) and Vector NTI software (InforMax Inc., North Bethesda, MD) was used to select appropriate primers near the transcriptional start site (Table 2). PCR was performed in a final volume of 50μl containing 100–200ng of bisulfite-treated DNA, 1× Qiagen PCR Buffer, 1.5mM MgCl2, 0.1mM of each dNTP, 0.2μM of each primer set, and 0.2U of HotStarTaq (Qiagen Inc., Mississauga, Ont., Canada). The amplifications consisted of an initial enzyme activation step at 95°C for 13min followed by 35 amplification cycles (94°C for 30s, annealing temperature for 30s, and 72°C for 45s) and a final incubation at 72°C for 5min. The annealing temperatures used were 58.9°C for the methylated TGFβRII primer set and 52.6°C for the unmethylated set. The methylation status of the hMLH1 and hMSH2 promoters was determined using published primer sets [6]. Normal bone-marrow DNA treated in vitro with SssI methyltransferase (New England Biolabs, Mississauga, Ont.) that methylates DNA at all CpG sites, served as the positive control for methylated TGFβRII and hMSH2 and DNA from the colorectal cancer cell line SW48 was used as a positive control for methylated hMLH1 promoter alleles. The specificity of MSP was confirmed by sequencing representative PCR products from each primer set on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) following purification using a Qiaquick PCR Purification Kit (Qiagen Inc., Mississauga, Ont.). 2.4. Analysis of hMSH2 and hMLH1 promoters for CpG methylation by TTGE CpG island methylation within hMSH2 and hMLH1 promoters was also determined by TTGE. In this analysis, the bisulfite-treated DNA is amplified with gene specific primers that amplify both unmethylated and methylated promoter sequences concurrently. Differentially methylated sequences within the PCR product are then resolved according to differences in melting temperatures. WinMelt software (Bio-Rad Laboratories Ltd., Mississauga, Ont.) was used to generate the melting profiles of unmethylated and methylated CpG promoter sequences. Vector NTI software (InforMax Inc., North Bethesda, MD) was used to select primers specific for bisulfite-treated DNA to amplify regions within CpG islands of hMSH2 (GenBank Primary Accession no. U23824) and hMLH1 (GenBank Primary Accession no. U83845) near the transcriptional starting points (Table 2). The primers were designed to contain the maximum number of thymidines converted from cytosines and to lack CpG dinucleotides in order to minimize amplification of genomic sequences not converted by bisulfite treatment and to avoid biased amplification of methylated alleles [20]. GC-clamps were included in the primer sequences to optimize separation of unmethylated and methylated alleles by TTGE. The PCR reactions consisted of an initial enzyme activation step of 13min at 95°C, followed by 40 amplification cycles (94°C for 30s, annealing temperature for 30s, and 72°C for 45s) and a final incubation at 72°C for 5min. The annealing temperatures used were 52.8°C for the hMSH2 primer set and 60.0°C for the hMLH1 set. PCR products were loaded onto 6% polyacrylamide gels containing either 7M (hMSH2) or 6M (hMLH1) urea. Gels specific for the hMSH2 PCR products were run at 130V for 7.0h in 1× Tris–acetate/EDTA buffer over a temperature range of 47–66°C at a 2.7°C/h ramping rate. Gels specific for the hMLH1 PCR products were run at 130V for 6.6h in 1× Tris–acetate/EDTA buffer over a temperature range of 48–58°C at a 1.5°C/h ramping rate. Following electrophoresis, gels were transferred and fixed to nylon membranes, pre-hybridized with oligonucleotide hybridization buffer and hybridized overnight to  -dCTP labeled oligonucleotide probes specific for hMSH2 sequences at 41°C or hMLH1 sequences at 45.5°C. 3. Results 3.1. Microsatellite mutations in TGFβRII (A)10 and caspase-5 (A)10 coding region repeats Frameshift mutations within the TGFβRII gene (A)10 repeat are a feature of MMR defective cancers and have been reported in patient biopsy specimens and cell lines from a variety of human solid tumors and in cell lines derived from human hematological cancers, including T-ALL [8], [9], [16], [17], [21]. TTGE analysis of the TGFβRII gene (A)10 repeat in human T-LBL/ALL identified frameshift mutations in three of 16 cases, all of which showed a mutant band migrating in the (A)9 position in addition to the wild-type (A)10 band. Sequencing confirmed that the mutant band represented deletion of one adenine within the (A)10 track (Fig. 1). TTGE analysis of control DNA showed the wild-type TGFβRII (A)10 band only in all 12 samples (data not shown). TTGE did not detect PCR artifacts often observed with sequencing gel methods [22], [23] as the (A)9, (A)10 and (A)11 plasmids showed only single bands migrating in the appropriate positions (Fig. 1). The melting profiles of the wild-type caspase-5 (A)10 repeat and mutant (A)9 and (A)11 repeats did not allow adequate differential resolution on TTGE gels. Therefore, a direct sequencing approach was used to screen for microsatellite mutations, with cloning and sequencing of representative screen positive cases carried out to confirm the occurrence of insertions or deletions within the caspase-5 (A)10 repeat. Using this approach, four of 16 human T-LBL/ALL cases but none of 12 control DNA samples screened positive for caspase-5 (A)10 microsatellite mutations as evidenced by overlapping sequences starting at the distal end of the repeat (Fig. 2). One of the positive cases also harbored a TGFβRII microsatellite mutation (Table 3). Cloning and sequencing of two screen positive cases confirmed the positive screening results (Fig. 2). In one case, mutant (A)9 sequences were identified in addition to wild-type (A)10 sequences and in the other case (A)9 and (A)11 were identified in addition to the (A)10 sequence. Overall, six of 16 (37.5%) T-LBL/ALL samples harbored microsatellite mutations in TGFβRII and/or caspase-5 and one case contained mutations in both repeats (Table 3). 3.2. TGFβRII promoter region methylation The region of the TGFβRII promoter and non-coding exon 1 is known to contain a CpG island and dense methylation in this region was recently identified in lung cancer cell lines [24]. We used MSP to investigate T-LBL/ALL for aberrant TGFβRII promoter region methylation in vivo and found four of 15 (26.7%) cases to contain methylated TGFβRII sequences, whereas no methylation was detected in any of 16 control samples (P<0.05). Direct sequencing of PCR products generated by primers specific for methylated sequences showed all CpG sites internal to the primers to be resistant to bisulfite conversion in the positive control and all four positive T-LBL cases. The corresponding unmethylated PCR products from the positive control and T-LBL cases were also sequenced and showed all CpG sites internal to the primers to be converted (Fig. 3). One of the four positive T-LBL cases also showed a TGFβRII (A)9 frameshift mutation (Table 3). 3.3. hMSH2 and hMLH1 promoter methylation Methylation of the hMLH1 promoter represents an important mechanism by which sporadic human colon, gastric and endometrial cancers acquire defective MMR [5], [6], [7], [25]. To investigate a possible relationship between the occurrence of TGFβRII and caspase-5 microsatellite mutations and methylation of MMR gene promoters in vivo, we analyzed T-LBL/ALL DNA following bisulfite treatment. Two cases (T1 and T14), neither of which harbored microsatellite mutations, could not be assessed for methylation due to insufficient available DNA. We first performed MSP using published primer sets designed to discriminate between methylated and unmethylated sequences present in the hMLH1 and hMSH2 promoters [6]. All 14 T-LBL/ALL samples we analyzed amplified with primers specific for unmethylated hMLH1 and hMSH2 only, whereas, the hMLH1 and hMSH2 positive controls amplified with primers specific for methylated sequences only (Fig. 4). While MSP remains the most common technique to determine methylation status, biased amplification of either methylated or unmethylated sequences is possible depending on the efficiency of the respective specific primers [20]. In order to exclude amplification bias as a cause of the observed lack of hMLH1 and hMSH2 promoter methylation by MSP, we designed a TTGE method that utilized hMLH1 and hMSH2 specific primers capable of simultaneously amplifying both unmethylated and methylated promoter sequences from bisulfite-treated DNA. This method resolves differentially methylated sequences within the hMLH1 and hMSH2 amplicons according to differences in melting temperatures and offers the additional advantage of allowing a large number of CpG sites to be assessed. TTGE analysis of both the hMLH1 and hMSH2 promoter regions, comprising 25 and 30 CpG sites, respectively, did not detect methylation in any of the 14 T-LBL/ALL samples. In contrast, hMLH1 and hMSH2 methylated positive controls showed predominantly methylated promoter sequences (Fig. 5; hMSH2 data not shown). Thus, TTGE results confirmed MSP studies and argue against amplification bias as a cause for the exclusively unmethylated hMLH1 and hMSH2 promoter sequences observed in T-LBL/ALL cases by MSP. 4. Discussion Analysis of human ALL cell lines and childhood ALL patient samples by Molenaar et al., found TGFβRII (A)10 and BAX (G)8 microsatellite mutations in the cell lines only, leading the investigators to speculate that in human hematological cancers, these changes may be selected for during the establishment of the cell lines [21]. However, as these microsatellite mutations were found in three of six adult ALL cell lines, but not in any of the six cell lines derived from children, an alternative explanation might be that MMR deficiency represents a pathogenetic event more important to the development of adult leukemia/lymphoma. Given this possibility, the compelling MSH2−/− murine model data implicating defective MMR in lymphoblastic tumor pathogenesis [13], and our previous study that identified hMSH2 coding region mutations in human adult T-LBL patient samples [12], we felt it was important to determine if coding region microsatellite mutations, characteristic of defective MMR, occur in these tumors in vivo. Our results identifying TGFβRII (A)10 and caspase-5 (A)10 mutations in adult T-LBL/ALL patient biopsy samples further implicates defective MMR in the pathogenesis of some of these tumors. The identification of TGFβRII (A)10 microsatellite mutations in T-LBL/ALL in vivo in this study corroborates previous investigations of human T-ALL cell lines [16], [17] and MSH2−/− murine lymphomas [13] and provides strong evidence for TGFβRII as a TSG in MMR defective T-LBL/ALL. The caspase-5 (A)10 repeat is a target of microsatellite mutations in MMR defective human solid cancers [11]. A recent cDNA array study identified capsase-5 as one of 12 genes with significantly reduced expression in highly metastatic subpopulations of an adenocarcinoma cell line [26]. This finding suggests that caspase-5 inactivation, like TGFβRII, BAX, and hMSH3, may have relevance to the pathogenesis of MMR defective cancers [11]. As well, Max, the central component of the Myc/Max/Mad network of transcription factors has now been identified as a caspase-5 substrate that is targeted for degradation during Fas-induced apoptosis in the human T-ALL cell line Jurkat [27]. That Max likely plays a key role in the apoptotic response of lymphoid cells, and in Jurkat cells appears to be regulated by caspase-5 activity, argues that the caspase-5 mutations detected in T-LBL/ALL may have pathogenetic significance. The identification of caspase-5 (A)10 microsatellite mutations in T-LBL/ALL in vivo is therefore, of interest because it strengthens the association with defective MMR and suggests a possible role for this gene as a tumor suppressor in these cancers. As other genetic and epigenetic mechanisms of inactivation may be possible, additional investigation is warranted to determine the extent to which caspase-5 is deregulated in T-LBL/ALL and to confirm its possible role as a TSG. We detected (A)9 deletion mutations in TGFβRII and both (A)9 deletion and (A)11 insertion mutations in caspase-5 using TTGE or direct sequencing as screening procedures. We used these approaches because sequencing gels can be associated with false positive results [22], [23]. This may reflect detection of amplicons larger and/or smaller than the wild-type product that are generated by the polymerase activity in vitro from unequal tailing or slippage. TTGE appears to be insensitive to polymerase artifacts as the TGFβRII control plasmids produced only single bands. Thus, TTGE may offer an improvement over sequencing gel methods provided the melting profiles of the wild-type and mutant repeat tracts permit adequate resolution. The direct sequencing procedure we used to analyze caspase-5 also offers the advantage of allowing the repeat tract to be directly examined for sequence abnormalities and is particularly useful for eliminating tailing artifacts as a source of false positives. However, the method is rather labor intensive and may not be suitable for large-scale analysis. Although, deletion mutations of mononucleotide repeats are reported more commonly than insertion mutations, the caspase-5 (A)11 mutations we detected have also been reported in human solid tumors in vivo [11] and are of particular interest because polymerase slippage error during PCR amplification is not felt to cause this change [22], [23]. In this regard, we are also confident that the (A)9 deletion mutations detected in TGFβRII and caspase-5 repeats are not the result of polymerase slippage artifact because we performed high fidelity amplification with Pfu, which is far less likely to introduce slippage errors than other polymerases, and we did not detect these mutations in control samples. In human solid cancer biopsy specimens and hematological cell lines, including T-ALL, target genes harboring frameshift microsatellite mutations frequently contain non-mutated alleles in addition to the mutant insertion or deletion allele(s) [11], [16], [17], [21]. Similar to these reports, the TGFβRII and caspase-5 frameshift mutations we identified in T-LBL/ALL in vivo were associated with non-mutated alleles. Of note, the TGFβRII frameshift mutations that occur at a high frequency in MSH2-/- murine T-LBL are associated with TGFβRII mRNA and protein loss, despite the consistent presence of non-mutated alleles [13]. In the HCT116 colon cancer cell line, one p16INK4A allele contains a coding region frameshift mutation and is expressed, whereas, the other allele is wild-type but is silenced due to selective hypermethylation of its promoter [28]. As the TGFβRII promoter region is known to contain a CpG island and dense methylation was recently found to occur in human lung cancer cell lines [24], we investigated T-LBL/ALL for aberrant TGFβRII methylation in vivo as a potential gene inactivating mechanism acting in concert with, or independently of, TGFβRII frameshift mutations. Although, we identified aberrant TGFβRII methylation in four of 15 (26.7%) T-LBL/ALL cases, only one of these cases (T5) also harbored a TGFβRII (A)10 frameshift mutation (Table 3). However, other genetic and epigenetic mechanisms of inactivation may be possible, including mutations outside of the (A)10 repeat as reported in colon cancer [29] and deacetylation of promoter region histones as reported in lung cancer [24]. Thus, additional investigation of the TGFβRII promoter and coding regions in T-LBL/ALL cases is warranted to determine the extent to which (A)10 frameshift mutations, other coding region mutations, promoter region methylation and alterations in chromatin structure may co-operate in vivo to deregulate TGFβRII. In contrast to the documented occurrence of hMLH1 and hMSH2 mutations in T-LBL/ALL there have been no reports of promoter methylation in either gene in this disease. We did not detect any hMLH1 or hMSH2 promoter methylation in the 14 T-LBL/ALL patient samples available for analysis, including all cases harboring microsatellite mutations, using MSP and our TTGE method that permits analysis of multiple CpG sites simultaneously and that reduces the potential for biased amplification of unmethylated or methylated sequences. That epigenetic silencing of these genes by promoter methylation may not be essential for the development of defective MMR is evidenced by previously reported studies. For example, the frequency of hMLH1 promoter methylation has been found to vary among types of MMR defective solid tumors with high frequencies reported in endometrial and sporadic colon cancers [5], [6], [7], [30], and no evidence of methylation in studies of pancreatic and head and neck cancer [30], [31]. As well, hMSH2 promoter methylation occurs very rarely in solid tumors [32], and in studies of MMR defective acute myeloid leukemia (AML) samples, loss of hMSH2 protein was found at a high frequency with no evidence of hMSH2 promoter methylation [33], [34]. Although, the number of cases we studied is small, our results strongly suggest that the acquisition of defective MMR, as evidenced by the occurrence of microsatellite mutations in defined target genes, is not the result of epigenetic silencing of hMLH1 or hMSH2. Further studies of T-LBL/ALL for mutations within the entire coding regions of hMLH1 and hMSH2, and for mutational or epigenetic inactivation of other MMR genes, such as hMSH6, may help define in vivo pathway(s) leading to defective MMR in human T-LBL/ALL [35], [36], [37]. Acknowledgements We thank Todd Reichert for assistance in preparing the figures and Dr. Bill DeCoteau for help in preparing the manuscript. This work was supported by Grants from the Medical Research Council of Canada, the National Cancer Institute of Canada and the Health Service Utilization and Research Commission of Saskatchewan. S. Scott provided the concept, design, collected and analyzed the data, drafted the article, and gave critical input to the revision. T. Kimura helped to collect the data, provide study materials and logistical support. R. Ichinohasama provided study materials. S. Bergen provided technical support. A. 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J. Med. Genet. 1999;36:678. MEDLINE a Saskatoon Cancer Centre, Department of Pathology, Royal University Hospital, University of Saskatchewan, 103 Hospital Drive, Saskatoon, SK, Canada S7N 0W8 b Department of Oral Pathology, Tohoku University, School of Dentistry, 4-1 Seiryo Machi, Aoba-Ku, Sendai 980-8575, Japan c Department of Pathology and Laboratory Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alta, Canada T2N 4N1  Corresponding author. Tel.: +1-306-655-2249; fax: +1-306-655-6791.
PII: S0145-2126(02)00078-4 © 2002 Elsevier Science Ltd. All rights reserved. | |
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