Affiliations of authors: Department of Pathology (OM, AS), Pediatrics (KS), and Community Health and Epidemiology (PP), Royal University Hospital and College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Correspondence to: Anurag Saxena, MBBS, MD(Path), FRCPC, Department of Pathology, Royal University Hospital, 103 Hospital Dr., Saskatoon, SK, Canada S7N 0W8 (e-mail: saxena{at}sask.usask.ca)
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ABSTRACT |
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INTRODUCTION |
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The MCL-1 gene is located on chromosome 1 and has three exons and two introns (12,13). Alternative splicing is responsible for generating at least two isoforms, antiapoptotic Mcl-1 and proapoptotic Mcl-1S (12,13). The structure of antiapoptotic Mcl-1 is similar to that of the Bcl-2 protein; it contains the Bcl-2 homology (BH) domains BH1, BH2, and BH3 and a C-terminal transmembrane region (14). Mcl-1 differs from Bcl-2, however, in that its N-terminal region has PEST sequences, which are responsible for rapid protein turnover (15,16). Mcl-1, similar to many other Bcl-2 family members, has the ability to interact with Bax, Bak, and Bcl-2 proteins (10,12,17).
The mechanisms that are responsible for altered expression of Mcl-1 in CLL have not been completely delineated. Mcl-1 is known to be regulated both transcriptionally and posttranscriptionally (11,13). Mcl-1 protein levels decline in spontaneously dying peripheral blood B cells (18) but are maintained in B cells cultured in the presence of survival signals, indicating their dependence on growth factors. The increased expression of Mcl-1 protein in CLL may depend on certain growth factors, including interleukin 4 (IL-4), IL-3, IL-13, and granulocytemacrophage colony-stimulating factor (GM-CSF) (11, 1824). Alterations in the MCL-1 gene promoter may affect transcriptional regulation by these extracellular signals. It is also possible that genetic changes in the MCL-1 gene may be directly responsible for altered expression of Mcl-1 protein, similar to what is seen with Bcl-2 (25,26) and Bax (27). Because rearrangements of chromosome 1, where the MCL-1 gene is located, are seen only rarely in CLL (28), it seems unlikely that cytogenetic abnormalities in this region are responsible for MCL-1 gene overexpression in CLL. We therefore sequenced the MCL-1 gene in CLL patients to identify any sequence abnormalities that might be associated with altered gene expression.
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SUBJECTS AND METHODS |
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This study was approved by the University of Saskatchewan Advisory Committee on Ethics in Human Experimentation. Fifty-eight CLL patients (median age = 69.5 years, range = 34 to 87 years) who were from Saskatoon Health Region hospitals and who were registered at the Saskatoon Cancer Center between 1980 and 2001 form the basis of this report. The samples for MCL-1 genetic studies were obtained from April 2000 through April 2001, which is the duration of the study approval. The patients included 25 women (median age = 71 years, range = 42 to 87 years) and 33 men (median age = 64 years, range = 34 to 84 years). Clinical information was obtained from patient charts in the Medical Records section of Saskatoon Cancer Center. CLL was diagnosed according to the revised National Cancer Institute and Revised European-American Lymphoma classification criteria (29,30). Eighteen healthy volunteers (seven women and 11 men, median age = 74.5 years, range = 36 to 91 years) served as control subjects.
At the time of enrollment in the study, 32 patients were in Rai stage 0 and 26 were in Rai stages IIV (four in stage I, nine in stage II, five in stage III, and eight in stage IV) (31) (Table 1). The disease was considered progressive when any of the following conditions were met: there was progression to a higher stage, there was transformation to lymphoma, the lymphocyte doubling time was less than 1 year, or the patient died because of the disease. According to these criteria, 13 patients were considered to have progressive disease. Five patients progressed to a higher stage during the course of this study: two from stage 0 to stage IV (patients 21 and 24), one from stage I to stage II (patient 33), one from stage I to stage IV (36), and one from stage II to stage IV (patient 44) (Table 1). In two patients, the disease transformed to lymphoma (patients 53 and 57). Five patients died as a result of the disease (patients 42, 47, 51, 56 and 58). One patient (patient 43) had a lymphocyte doubling time of less than 1 year as the only defining feature of progression. Of the 58 patients, 12 died during the course of this study, eight because of CLL (patients 24, 36, 42, 47, 51, 56, 57, and 58) and four because of unrelated causes (patients 13, 15, 35, and 54).
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Lymphocyte and B-Cell Separation
Blood samples (24 ml) for MCL-1 genetic changes were obtained from all 22 treated patients, all 36 untreated patients, and 18 healthy volunteers. Blood samples were stored at 4 °C for 35 hours, after which lymphocytes were separated by Ficoll-Hypaque gradient centrifugation using Histopaque-1077 (Sigma, St. Louis, MO). Blood was diluted (1:1) with phosphate-buffered saline (PBS), and the diluted blood sample was layered on Histopaque and centrifuged at 400g for 30 minutes at room temperature. Lymphocytes were collected, washed twice with PBS, and resuspended in 200 µL PBS. Cells for genetic studies were stored at 70 °C. Protein and RNA were extracted immediately after lymphocyte separation using TRIzol, and protein and RNA samples were stored at 70 °C. Two lymphoma cell lines, RL and BC-3, were used as additional controls.
In addition, magnetic sorting was used to obtain a purified B-cell (CD20-positive) population from the peripheral blood lymphocytes of three additional healthy volunteers. This purified B-cell population was used as an additional control in the DNA sequence analyses only, because mononuclear population from whole blood contains less than 10% B cells in normal healthy individuals. CD20-positive cells were separated using midi separation columns, anti-PE micro beads, and MACS magnet (Miltenyi Biotech, Auburn, CA) according to the manufacturer's instructions. This procedure was performed at the Flow Cytometry facility of the Vaccine and Infectious Disease Organization in Saskatoon, Saskatchewan. The purity of this B-cell population was checked by flow cytometry, and the population was found to be 93%97% pure. DNA, RNA, and protein were extracted from the mononuclear cells both before and after B-cell purification.
Cell Lines
The RL (CRL-2261) and BC-3 (CRL-2277) lymphoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured at 37 °C under 5% CO2 in RPMI-1640 medium (Gibco BRL, Grand Island, NY) with 10% fetal bovine serum (Gibco BRL) for RL and 20% fetal bovine serum for BC-3 cells. Cells were pelleted by centrifugation at 400g for 10 minutes and washed twice with PBS, and 510 x 106 cells were resuspended in 200 µL PBS.
DNA Sequence Analysis
Genomic DNA was extracted from samples by using the QIAamp Tissue Kit (Qiagen, Mississauga, ON) according to the manufacturer's instructions. The blood and body fluid spin protocol of the QIAamp Tissue kit was used to purify DNA from lymphocytes, purified B cells, and the two cell lines. Two normal tissue samples (one from breast and one from thyroid from two different CLL patients) were obtained from paraffin-embedded tissue, and the tissue protocol of the QIAamp Tissue kit was used to extract DNA from these samples. All three exons, the first and second introns, and the flanking 5' and 3' regions of the MCL-1 gene were amplified with the polymerase chain reaction (PCR) using 13 sets of primers. DNA quantity and quality were determined using a spectrophotometer, and samples were diluted with PCR-grade water to 250 ng/µL. DNA was amplified in a 50-µL reaction mix containing 2 pM of each primer and 25 µL of Ready Mix RedTaq PCR Reaction Mix (Sigma, St. Louis) that included 20 mM TrisHCl, 100 mM KCl, 3 mM MgCl2, 0.4 mM dNTP, and 1.5 units of Taq DNA polymerase. Thirty-eight cycles of amplification (denaturation at 94 °C for 1 minute, annealing at 55 °C for 2 minutes and an extension at 72°C for 3 minutes, with the initial denaturation at 95 °C and a final extension at 72 °C for 5 minutes) were performed in a PTC-200 Thermocycler (MJ Research, Waltham, MA). A negative control was run in each experiment that consisted of all reagents except DNA. PCR products were electrophoresed on 1.6% agarose gels, visualized by staining with ethidium bromide, cut out of the gel, and purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The PCR products were subject to direct sequencing on a 377 ABI Prism DNA Sequencer (Perkin Elmer, Foster City, CA). The sequences were compared with the MCL-1 data in the GenBank (accession numbers AF198614, AF162677, AF162676, AF118124, NM_021960, and L08246). Putative transcription factor binding sites in the promoter region were identified using TFSearch (32).
RNA Analysis
RNA was extracted from isolated lymphocytes, purified B cells, and cell lines using TRIzol (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. RNA was quantified using a spectrophotometer and stored at 70 °C. Gene expression at the mRNA level was analyzed by ribonuclease (RNase) protection assay. Ten micrograms of RNA from each sample was prepared using the In Vitro Transcription kit (Pharmingen, Mississauga, ON) and was hybridized with the labeled multiprobe template set template hApo-2c (Pharmingen). Each gel included a positive control (HeLa control RNA, supplied with the kit) and a negative control (yeast tRNA, supplied with the kit). The products were resolved on a 5% denaturing polyacrylamide gel, dried, and autoradiographed. Bands were analyzed and quantified using Molecular Imager FX and Quantity One software (Bio-Rad, Mississauga, Canada). Tests were performed in duplicate, and two values for each sample were averaged. The housekeeping gene product L32 served as a control for equal loading of samples. The level of each mRNA sample was normalized against the level of L32, and relative expression levels were calculated.
Protein Analysis
Western blot analysis of cell lysates was performed as previously described (8). The housekeeping gene product GAPDH, which was detected with anti-GAPDH mouse monoclonal antibodies [Novus Biologicals, Littleton, CO], served as a loading control (8). Ten micrograms of cell lysates were run on 12% SDSpolyacrylamide gels and blotted onto nitrocellulose filters using a semidry transfer cell (Bio-Rad). Filters were pretreated with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% nonfat dry milk and incubated at 4 °C overnight in TBS buffer (TBS plus 0.1% Tween-20, 0.5% nonfat dry milk, and 0.1% fetal bovine serum) containing primary (1:500) anti-Mcl-1 antibody (mouse monoclonal, NeoMarkers, Fremont, CA). Blots were washed and incubated for 1 hour at room temperature with goat anti-mouse immunoglobulin G (1:2000) antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive material was detected by the enhanced chemiluminescence method (NEN, Life Science Products, Woodbridge, ON, Canada) according to the manufacturer's instructions. Protein levels for each sample were normalized against the corresponding GAPDH level and measured using Quantity One, version 2.4 software (Bio-Rad) (8). The western blot analysis was performed in duplicate, the relative expression levels normalized to GAPDH levels were calculated for each replicate, and the average value was used for statistical analysis.
Biochemical and Flow Cytometric Parameters
Lactate dehydrogenase (LDH) levels were determined for 28 patients by automated measurement of NADH based on lactate to pyruvate enzymatic conversion using Synchron LX 20 (Beckman Coulter, Fullerton, CA) as described (33). Expression data for CD38, a known prognostic factor for CLL, were available for 38 CLL patients from flow cytometric analysis performed in the diagnostic laboratory. For this analysis, dual-parameter (CD19/CD38) histograms were used to determine CD38 expression in CD19-positive CLL cells. A 30% cut-off point was used; that is, the samples were considered CD38 positive if this antigen was present in 30% or more tumor cells and negative if expression was present in less than 30% cells (3436).
Statistical Analysis
Statistical analysis was performed to determine the statistical significance of differences between two groups of patients (those with and those without nucleotide sequence insertions in the MCL-1 promoter) in mRNA and protein expression levels, Rai stage, CD38 expression, LDH levels, total white cell count, sex, age, treatment response, and survival. Because the data were not distributed normally, medians and interquartile ranges (IQRs) were used to describe the distributions of continuous variables. The nonparametric MannWhitney U test was used to determine the statistical significance of differences in median values. The chi-square test was used to test the association between categorical variables and the presence and absence of promoter insertions. Fisher's exact test was used if the expected frequency of an event was less than 5 in any cell of a 2 x 2 table. All statistical tests were two-sided.
Patient survival was analyzed using KaplanMeier survival curves. Overall and disease-specific survivals with 95% confidence intervals (CI) are reported. The outcome of interest was death, and the time to death was the survival time (calculated in months from the diagnosis to the time of death). For disease-specific survival analysis, subjects were censored if they were still alive at the time of analysis or if the death was not associated with CLL or its treatment. For overall survival analysis, subjects were censored only if they were still alive at the time of analysis of data. The survival time distributions for two groups (with and without promoter insertions) were compared using the log-rank test.
Cox proportional hazards analysis was used to determine the effect of prognostic factors on survival. For this analysis, Rai stage was grouped into two categories: low- and intermediate-risk group (Rai stages 0, I, and II) and advanced-risk group that combine patients with anemia or thrombocytopenia (Rai stages III and IV) (35). Results are presented as hazard ratios and 95% CIs. The proportionality assumption was satisfied when tested using log minus log test.
P values were considered statistically significant at P = .05 or less. Statistical analysis was performed using SPSS software, version 11 (Chicago, IL).
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RESULTS |
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We purified DNA from the lymphocytes of 58 patients with CLL and 18 control subjects, from a purified B-cell population from three additional controls, from two normal tissue samples from CLL patients, and from two lymphoma cell lines (RL and BC-3). In initial analyses, primer sets 1 and 313 were used to sequence the gene in samples from the first 20 consecutive CLL patients, eight control subjects, and the two cell lines to identify possible sites of interest. No mutations or polymorphisms were detected in the open reading frame, the two introns, or the 3' untranslated region of the gene. We found six novel sequence changes in the 5' region of the gene in all 20 patients, in four of eight control subjects, and in both cell lines. These novel findings included four single nucleotide polymorphisms (SNPs) (in 16 patients, four control subjects, and the RL cell line) or short sequence insertions, either 6 or 18 nucleotides, in the MCL-1 promoter region (in five patients and the BC-3 cell line).
The SNPs were as follows: G(298)C (seen in 16 of the 20 CLL patients, of whom 11 were heterozygous and five were homozygous; in four of eight control subjects, all of whom were heterozygous; and in the RL cell line, which was homozygous); C(236)A (seen in the same 16 CLL patient samples, of whom five were heterozygous and eleven homozygous; in four of eight control subjects, all of whom were heterozygous; and in the RL cell line, which was homozygous); A(528)C (seen in 10 of 19 CLL patients [for one patient a part of the sequence was not readable], in two control subjects, and in RL cell line; all of which were homozygous); and C(160)G in a heterozygous state in one CLL patient and in the BC-3 cell line.
Based on these results, we used primer set 2 to sequence the 6- or 18-nucleotide insertion bearing part of the promoter in all samples. All sequences from the control subjects consisted of five GGCCCC repeats and one GCTCA motif located after the first GGCCCC motif in the promoter region of the MCL-1 gene. This region was amplified by primer set 2 (spanning 311 to 94 bp) upstream from the major transcription site. In 11 of 58 CLL patients, we found an 18-nucleotide insertion, GGCTCAGGCCCCGGCCCC, representing two extra GGCCCC repeats and one extra GGCTCA motif in the MCL-1 promoter. Five patients were heterozygous for the insertion, and six were homozygous (Table 1). In addition, in six of the remaining 47 patients and in the BC-3 cell line, we found a 6-nt insertion (GGCCCC) that represents one extra repeat. Four patients were heterozygous, and two patients and the BC-3 cell line were homozygous. In all cases, the 18- and 6-nucleotide insertions were inserted at the 190-bp position upstream of the major transcription start site (GenBank, AN: AF198614) (Fig. 1B). These insertions were not identified in the normal tissue from CLL patients, both of whose peripheral blood lymphocytes harbored these insertions. These insertions were also not present in any of the control samples from healthy volunteers. Both insertions were considered together as one group for statistical analysis because the numbers in either category (18- or 6-nucleotide) were too small to analyze alone.
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Association Between 5'-Region Insertion and Higher Mcl-1 mRNA Levels
We determined Mcl-1 mRNA levels by RNase protection assay in 20 consecutive CLL patients with a sample amount sufficient for both RNA and DNA extraction (Fig. 2). The analyzed data did not have a normal distribution; therefore, nonparametric tests were used. Levels of Mcl-1 mRNA (relative to that of L32) did not differ statistically significantly (P = .573, U test) between CLL patients (median = 11.9 relative units, IQR = 5.1 to 19.4) and control samples from healthy volunteers (median = 11.4 relative units, IQR = 5.7 to 15.0). Within the CLL group, however (n = 20), samples bearing the insertion (n = 4, all with the 18-nt insertion) had statistically significantly (P = .030, U test) higher levels of MCL-1 mRNA (median = 26.8 relative units. IQR = 14.9 to 35.2) than samples without (n = 16) the insertion (median = 8.8 relative units, IQR = 3.9 to 15.7) (Fig. 2). None of the four samples with insertions had any SNPs; however, three patients without an insertion had SNPs in their promoters. In addition, the BC-3 cell line had higher mRNA levels (9.6 relative units) than the RL cell line (6.1 relative units).
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We next carried out western blot analysis to determine whether the promoter insertions had an effect on Mcl-1 protein levels (Fig. 3). The level of the 41-kDa Mcl-1 protein was slightly higher in 18 consecutive patients with sample amounts sufficient for nucleic acid and protein extraction (median = 0.63 relative units, IQR = 0.40 to 0.81) than in control subjects (median = 0.41 relative units, IQR = 0.19 to 0.45), although the difference was not statistically significant (P = .358, U test). Again, the analyzed data were not distributed normally; therefore, nonparametric tests were used for statistical analysis. Within the CLL group (n = 18), samples bearing the insertion (n = 6; four with a 18-nt and two with a 6-nt insertion) had statistically significantly (P = .021, U test) higher levels of Mcl-1 protein (median = 0.84 relative units, IQR = 0.81 to 1.0) than those (n = 12) without the insertion (median = 0.47 relative units, IQR = 0.32 to 0.70) (Fig. 3). One patient with an insertion and one without an insertion had SNPs in their promoters. In addition, the BC-3 cell line had higher Mcl-1 protein levels (0.94 relative units) than the RL cell line (0.67 relative units).
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We next examined the association of the MCL-1 6-nt or 18-nt insertion with Rai stage and clinical response. One of the two insertions was present in 14 of the 45 (31%) patients with Rai stages 0, I, or II disease and in three of the 13 (23%) patients with stage III or IV disease (Table 2) (P = .736, Fisher's exact test). An insertion was present in seven of the 10 (70%) patients who had no response to treatment and was absent in all 12 (100%) patients who had a complete or partial response (P = .001, Fisher's exact test) (Table 2). There was no association between the size (6 or 18 nt) of the insertion and Rai stage, disease progression, or treatment response (data not shown). There was also no association of the any of the single-nucleotide polymorphisms in the MCL-1 promoter with Rai stage (data not shown).
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We next examined the association between the MCL-1 insertion and known demographic and prognostic factors for CLL: LDH levels, white blood cell counts, sex, age, and CD38 expression. LDH levels were available for 28 patients. Levels were not statistically significantly different in the 10 patients with either (6- or 18-nt) insertion (median = 142 U/ L, IQR = 124 to 377) and the 18 patients without the insertion (median = 162.5 U/ L, IQ: 159 to 177) (P = .430, U test). Two patients with an insertion and five without an insertion had SNPs in their promoters. Total white cell count at diagnosis was available for 56 CLL patients and was not statistically significantly different between patients with an insertion (n = 17) (median = 35 x 109/L, IQ: 14.3 to 66.9) and those without (n = 39) (median = 28.6 x 109/L, IQ: 19.9 to 58.2) (P = .780, U test). The male to female ratio was similar in patients with and without an insertion; the group with an insertion included seven women and 10 men (ratio = 1:4), and the group with no insertion included 18 women and 23 men (ratio = 1:3) (P = .849, chi-square test) (Table 2). The median age was also similar in the two groups (median = 71 years, range = 52 to 82 years for those with an insertion; median = 69 years, range = 34 to 87 years for those without an insertion).
We had data on expression of the negative prognostic factor CD38 in 38 patients. CD38 expression varied from 2% to 89.9% (median = 21.7%, IQR = 5.9 to 44.5). With respect to the 30% cut-off point (see "Methods" section), 16 patients (42.1%) were CD38-positive and 22 (57.9%) patients were CD38-negative. Only one of these 38 patients died of CLL-related death at the time of data analysis. Therefore, the disease-specific survival median identified by KaplanMeier survival curve analysis was not reached in either CD38-positive or CD38-negative groups and did not differ significantly (data not shown).
Of the 21 CD38-negative patients, 10 had a promoter insertion and 11 did not. Of the 17 CD38-positive patients, one had a promoter insertion and 16 did not (P = .0099, Fisher's exact test). CD38 expression was statistically significantly lower (P = .025, U test) in the 11 patients with an insertion (median = 7.7%, IQR = 5.4 to 17.8) than in the 27 patients without an insertion (median = 40.9%, IQR = 7.9 to 57.7).
To determine if the MCL-1 promoter insertion is a prognostic factor, we developed a Cox model that included the known prognostic factors: Rai stage, white blood cell count, LDH, CD38 expression, and age, along with MCL-1 insertion status. Although the hazard ratios were high (above 1.0) for CD38 and LDH, they were not statistically significant (P>0.05). Statistically significant P values were obtained for the stage and insertion status values only; therefore, all other values were excluded from the final Cox model. Patients in the advanced Rai stage group had a statistically significantly higher risk of dying from CLL (HR = 12.3, 95% CI = 2.2 to 68.8) than those in the low/ intermediate-risk group (P = .004). After adjustment for Rai stage, patients with an insertion were at higher risk of dying from CLL (HR = 54.7, 95% CI = 4.7 to 634) than those without the insertion (P = .001). The wide confidence intervals probably reflect the small sample size. The large number of possible prognostic factors and the small number of events lead to a danger of overfitting the model and also result in underpowering the analysis.
Association of MCL-1 Insertion With Disease Progression and Survival
We next examined the association of the presence of an insertion with disease progression and overall and disease-specific survival in the entire group of patients using KaplanMeier survival analysis. The disease progressed rapidly (within 2 years after diagnosis) in eight of 17 (47%) patients with one of the promoter insertions and in five of 41(12%) patients without an insertion (P = .012, chi-square exact test). Both overall (Fig. 4, A) and disease-specific (Fig. 4, B) survival were statistically significantly different in patients with and without the insertion (P = .009 and P<.001, respectively, log-rank test). Survival of patients with the insertion ranged from 5 to 90 months (median of 90 months), whereas the survival of patients without the insertion ranged from 1 to 249 months (median not yet reached) (Fig. 4).
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DISCUSSION |
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In all cases, the inserted sequence was located at position 190 upstream of the major transcription site of the MCL-1 gene. This was in the GC-rich region of the MCL-1 promoter, which in DNA from control subjects contains five (GGCCCC) repeats and one GCTCA motif immediately downstream of the first GGCCCC motif; the inserted sequences represent extra repeats (GGCCCC and GGCTCA). The chromatin in this region has an "open" configuration, with no nucleosomes or histone H1, which makes the DNA accessible to transcription factors (37). Indeed, this region contains a putative binding site for several transcription factors (32), including Sp1. Multimerization of the Sp1 binding site or experimental addition of an extra Sp1 site is known to lead to superactivation of genes with Sp1 binding sites in their promoters (3739). Moreover, this region of the Mcl-1 promoter where an insertion was found is also a downstream target of GM-CSF, an antiapoptotic regulator of Mcl-1 signaling (40) that stimulates MCL-1 expression mainly at the transcriptional level (11,41). Additional evidence of the importance of the region in which the insertion is found is provided by a functional study (41); U937 macrophage/monocyte cells transfected with an MCL-1 promoter-reporter gene construct lacking a 47-bp region (including six GGCCCC repeats and two GCTCA motifs) of the promoter had statistically significantly lower GM-CSFdependent activation of MCL-1 expression than cells transfected with a MCL-1 promoter-reporter gene construct containing the 47-bp region in the MCL-1 promoter.
Our results show that MCL-1 promoter insertion led to higher (by 1.8-fold) protein (P = .021) and mRNA (by threefold) (P = .030) expression in CLL patients with the insertion compared with those lacking it (Figs. 2 and 3). In addition, we compared Mcl-1 mRNA and protein levels in two lymphoma cell lines: RL, which does not have an insertion, and BC-3, which contains a 6-nt insertion. The presence of the extra GGCCCC repeat in BC-3 cell line was associated with an approximately 50% increase in mRNA and 40% increase in protein levels compared with levels in RL cell line.
The promoter insertions we identified have clinical implications. Specifically, the disease progressed more rapidly in patients with an insertion compared with those without an insertion (P = .012), and patients with an insertion were more likely to fail to respond to standard chlorambucil and prednisone chemotherapy than those without an insertion (P = .001). In addition, patients with an insertion had statistically significantly shorter overall and disease-specific survival than the patients without an insertion (median 90 months versus median not reached, P = .0088 and P<.001).
Levels of two markers that are prognostic for CLL, white blood cell count and LDH, were not statistically significantly different in patients with and without an insertion. In addition, CD38 expression (using the standard 30% cutoff point) did not have prognostic significance when evaluated by either Cox regression or the KaplanMeier method (data not shown). However, our ability to carry out CD38 analyses was limited because CD38 expression values were not available for all patients enrolled in the study. Moreover, the patients for whom this parameter was available in the present study did not reach previously reported disease-specific survival medians, 79 to 90 months for CD38-positive patients and 113 months to "not reached" for CD38-negative patients (36,42). Multivariable analysis in a larger group of patients is needed to establish the prognostic significance of known prognostic factors in relation to the genetic changes in the MCL-1 gene. It will also be important to carry out a multivariable analysis that includes other factors recently shown to be of prognostic importance in CLL, including ZAP70 (43), VH gene mutational status (44), p53 mutational status (44), and cytogenetics (42).
Although CD38 was not of prognostic importance in the present work, we found that 10 of the 11 patients with a promoter insertion for whom CD38 information was available were also CD38-negative. It seems possible that the presence of a promoter insertion separates a group of patients with worse prognosis out of CD38-negative CLL patients, who have a good prognosis overall. One mechanism by which this could arise is if the presence of MCL-1 promoter insertions associated with increased Mcl-1 mRNA and protein expression contributes to the resistance to apoptosis of CD38-negative tumor cells.
In addition to detecting 6- and 18-nt insertions in the MCL-1 promoter, we identified SNPs in the promoter. These SNPs may also be involved in the transcriptional regulation of MCL-1 simply because of their location; for instance, C(-236)A SNP is present in the binding site of the cell cycle regulatory transcription factor CRE-BP (45,46). The possibility that SNPs may alter Mcl-1 protein regulation needs to be evaluated in future studies.
This study presents novel genetic findings in a small number of CLL patients and has several limitations. These include small sample size for mRNA and protein analysis, CD38 expression, and LDH levels. A large number of known prognostic markers and relatively small number of deaths, makes it impossible to include all factors into the Cox model without danger of overfitting it. These issues should be addressed in future studies.
In conclusion, we have reported a novel 6- or 18-nt insertion in the promoter of MCL-1 gene of CLL patients associated with mRNA and protein overexpression, rapid disease progression, and failure to respond to chemotherapy. The increased expression of the MCL-1 gene may be due to the 6- or 18-nt insertion in the region known to be a binding site for GM-CSF, the Mcl-1 signaling pathway activator. It is also possible that the size of the insertion, 6- versus 18-nt, has an effect on mRNA and protein expression and prognosis; this question needs to be addressed in a larger group of patients.
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NOTES |
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Manuscript received May 29, 2003; revised March 2, 2004; accepted March 9, 2004.
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